US20040115215A1

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Publication number: US20040115215A1
Application number: US10/729,122
Authority: US
Inventors: James Williams
Original assignee: Allergan Sales LLC
Current assignee: Allergan Sales LLC
Priority date: 1995-03-16
Filing date: 2003-12-05
Publication date: 2004-06-17
Also published as: US6967088B1; US20040219637A1; US20040142455A1; US20050238665A1; US20070269861A1; US20030219457A1; US20040235118A1; US20030215468A1; US20040253673A1

Abstract

The present invention includes recombinant proteins derived fromClostridium botulinumtoxins. In particular, soluble recombinantClostridium botulinumtype A, type B and type E toxin proteins are provided. Methods which allow for the isolation of recombinant proteins free of significant endotoxin contamination are provided. The soluble, endotoxin-free recombinant proteins are used as immunogens for the production of vaccines and antitoxins. These vaccines and antitoxins are useful in the treatment of humans and other animals at risk of intoxication with clostridial toxin.

Description

This application is a Continuation-In-Part of copending application Ser. No. 08/405,496, filed Mar. 16, 1995.[0001]

FIELD OF THE INVENTION

The present invention relates to the isolation of polypeptides derived from[0002]Clostridium botulinumneurotoxins and the use thereof as immunogens for the production of vaccines, including multivalent vaccines, and antitoxins.

BACKGROUND OF THE INVENTION

The genus Clostridium is comprised of gram-positive, anaerobic, spore-forming bacilli. The natural habitat of these organisms is the environment and the intestinal tracts of humans and other animals. Indeed, clostridia are ubiquitous; they are commonly found in soil, dust, sewage, marine sediments, decaying vegetation, and mud. [See e.g. P. H. A. Sneath el al., “[0003]Clostridium,” Bergey's Manual® of Systematic Bacteriology, Vol. 2, pp. 1141-1200, Williams & Wilkins (1986).] Despite the identification of approximately 100 species of Clostridium, only a small number have been recognized as etiologic agents of medical and veterinary importance. Nonetheless, these species are associated with very serious diseases, including botulism, tetanus, anaerobic cellulitis, gas gangrene, bacteremia, pseudomembranous colitis, and clostridial gastroenteritis. Table 1 lists some of the species of medical and veterinary importance and the diseases with which they are associated. As virtually all of these species have been isolated from fecal samples of apparently healthy persons, some of these isolates may be transient, rather than permanent residents of the colonic flora.

TABLE 1


Clostridium Species Of Medical And Veterinary Importance*

Species	Disease

C. aminovalericum	Bacteriuria (pregnant women)
C. argentinense	Infected wounds; Bacteremia; Botulism; Infections of amniotic
	fluid
C. baratii	Infected war wounds; Peritonitis; Infectious processes of the eye,
	ear and prostate
C. beijerinckikii	Infected wounds
C. bifermentans	Infected wounds; Abscesses; Gas Gangrene; Bacteremia
C. botulinum	Food poisoning; Botulism (wound, food, infant)
C. butyricum	Urinary tract, lower respiratory tract, pleural cavity, and
	abdominal infections; Infected wounds; Abscesses; Bacteremia
C. cadaveris	Abscesses; Infected wounds
C. carnis	Soft tissue infections; Bacteremia
C. chauvoei	Blackleg
C. clostridioforme	Abdominal, cervical, scrotal, pleural, and other infections;
	Septicemia; Peritonitis; Appendicitis
C. cochlearium	Isolated from human disease processes, but role in disease
	unknown.
C. difficile	Antimicrobial-associated diarrhea; Pseudomembranous
	enterocolitis; Bacteremia; Pyogenic infections
C. fallax	Soft tissue infections
C. ghnoii	Soft tissue infections
C. glycolicum	Wound infections; Abscesses; Peritonitis
C. hastiforme	Infected war wounds; Bacteremia; Abscesses
C. histolyticum	Infected war wounds; Gas gangrene; Gingival plaque isolate
C. indolis	Gastrointestinal tract infections
C. innocuum	Gastrointestinal tract infections; Empyema
C. irregulare	Penile lesions
C. leptum	Isolated from human disease processes, but role in disease
	unknown.
C. limosum	Bacteremia; Peritonitis; Pulmonary infections
C. malenominatum	Various infectious processes
C. novyi	Infected wounds; Gas gangrene; Blackleg, Big head (ovine);
	Redwater disease (bovine)
C. oroticum	Urinary tract infections; Rectal abscesses
C. paraputrificum	Bacteremia; Peritonitis; Infected wounds; Appendicitis
C. perfringens	Gas gangrene; Anaerobic cellulitis; Intra-abdominal abscesses;
	Soft tissue infections; Food poisoning; Necrotizing pneumonia;
	Empyema; Meningitis; Bacteremia; Uterine Infections; Enteritis
	necrotans; Lamb dysentery; Struck; Ovine Enterotoxemia;
C. putrefaciens	Bacteriuria (Pregnant women with bacteremia)
C. putrificum	Abscesses; Infected wounds; Bacteremia
C. ramosum	Infections of the abdominal cavity, genital tract, lung, and biliary
	tract; Bacteremia
C. sartagoforme	Isolated from human disease processes, but role in disease
	unknown.
C. septicum	Gas gangrene; Bacteremia; Suppurative infections; Necrotizing
	enterocolitis; Braxy
C. sordellii	Gas gangrene; Wound infections; Penile lesions; Bacteremia;
	Abscesses; Abdominal and vaginal infections
C. sphenoides	Appendicitis; Bacteremia; Bone and soft tissue infections;
	Intraperitoneal infections; Infected war wounds; Visceral gas
	gangrene; Renal abscesses
C. sporogenes	Gas gangrene; Bacteremia; Endocarditis; central nervous system
	and pleuropulmonary infections; Penile lesions; Infected war
	wounds; Other pyogenic infections
C. subterminale	Bacteremia; Empyema; Biliary tract, soft tissue and bone
	infections
C. symbiosum	Liver abscesses; Bacteremia; Infections resulting due to bowel
	flora
C. tertium	Gas gangrene; Appendicitis; Brain abscesses; Intestinal tract and
	soft tissue infections; Infected war wounds; Periodontitis;
	Bacteremia
C. tetani	Tetanus; Infected gums and teeth; Corneal ulcerations; Mastoid
	and middle ear infections; Intraperitoneal infections; Tetanus
	neonatorum; Postpartum uterine infections; Soft tissue infections,
	especially related to trauma (including abrasions and lacerations);
	Infections related to use of contaminated needles
C. thermosaccharolyticum	Isolated from human disease processes, but role in disease
	unknown.


#Toxins, 2d ed., pp. 66-67, American Society for Microbiology (1986); R. Berkow and A. J. Fletcher (eds.), “Bacterial Diseases,” Merck Manual of Diagnosis and Therapy, 16th ed., pp. 116-126, Merck Research Laboratories, Rahway, N.J. (1992); and O. H. Sigmund and C. M. Fraser (eds.), “Clostridial Infections,” Merck Veterinary Manual,
#5th ed., pp. 396-409, Merck & Co., Rahway, N.J. (1979).

In most cases, the pathogenicity of these organisms is related to the release of powerful exotoxins or highly destructive enzymes. Indeed, several species of the genus Clostridium produce toxins and other enzymes of great medical and veterinary significance. [C. L. Hatheway, Clin. Microbiol. Rev. 3:66-98 (1990).][0004]

Perhaps because of their significance for human and veterinary medicine, much research has been conducted on these toxins, in particular those of[0005]C. botulinumandC. difficile.

[0006]C. botulinum

Several strains of[0007]Clostridium botulinumproduce toxins of significance to human and animal health. [C. L. Hatheway, Clin. Microbiol. Rev. 3:66-98 (1990)] The effects of these toxins range from diarrheal diseases that can cause destruction of the colon, to paralytic effects that can cause death. Particularly at risk for developing clostridial diseases are neonates and humans and animals in poor health (e.g., those suffering from diseases associated with old age or immunodeficiency diseases).

[0008]Clostridium botulinumproduces the most poisonous biological toxin known. The lethal human dose is a mere 10⁻⁹mg/kg bodyweight for toxin in the bloodstream. Botulinal toxin blocks nerve transmission to the muscles, resulting in flaccid paralysis. When the toxin reaches airway and respiratory muscles, it results in respiratory failure that can cause death. [S. Arnon, J. Infect. Dis. 154:201-206 (1986)]

[0009]C. botulinumspores are carried by dust and are found on vegetables taken from the soil, on fresh fruits, and on agricultural products such as honey. Under conditions favorable to the organism, the spores germinate to vegetative cells which produces toxin. [S. Arnon, Ann. Rev. Med. 31:541 (1980)]

Botulism disease may be grouped into four types, based on the method of introduction of toxin into the bloodstream. Food-borne botulism results from ingesting improperly preserved and inadequately heated food that contains botulinal toxin. There were 355 cases of food-borne botulism in the United States between 1976 and 1984. [K. L. MacDonald et al., Am. J. Epidemiol. 124:794 (1986).] The death rate due to botulinal toxin is 12% and can be higher in particular risk groups. [C. O. Tacket et al., Am. J. Med. 76:794 (1984).] Wound-induced botulism results from[0010]C. botulinumpenetrating traumatized tissue and producing toxin that is absorbed into the bloodstream. Since 1950, thirty cases of wound botulism have been reported. [M. N. Swartz, “Anaerobic Spore-Forming Bacilli: The Clostridia,” pp. 633-646, in B. D. Davis et al., (eds.),Microbiology,4th edition, J. B. Lippincott Co. (1990).] Inhalation botulism results when the toxin is inhaled. Inhalation botulism has been reported as the result of accidental exposure in the laboratory [E. Holzer, Med. Klin. 41:1735 (1962)] and could arise if the toxin is used as an agent of biological warfare [D. R. Franz et al., inBotulinum and Tetanus Neurotoxins, B. R. DasGupta, ed., Plenum Press, New York (1993), pp. 473-476]. Infectious infant botulism results fromC. botulinumcolonization of the infant intestine with production of toxin and its absorption into the bloodstream. It is likely that the bacterium gains entry when spores are ingested and subsequently germinate. [S. Arnon, J. Infect. Dis. 154:201 (1986).] There have been 500 cases reported since it was first recognized in1976. [M. N. Swartz, supra.]

Infant botulism strikes infants who are three weeks to eleven months old (greater than 90% of the cases are infants less than six months). [S. Arnon, J. Infect. Dis. 154:201 (1986).] It is believed that infants are susceptible, due, in large part, to the absence of the full adult complement of intestinal microflora. The benign microflora present in the adult intestine provide an acidic environment that is not favorable to colonization by[0011]C. botulinum. Infants begin life with a sterile intestine which is gradually colonized by microflora. Because of the limited microflora present in early infancy, the intestinal environment is not as acidic, allowing forC. botulinumspore germination, growth, and toxin production. In this regard, some adults who have undergone antibiotic therapy which alters intestinal microflora become more susceptible to botulism.

An additional factor accounting for infant susceptibility to infectious botulism is the immaturity of the infant immune system. The mature immune system is sensitized to bacterial antigens and produces protective antibodies. Secretory IgA produced in the adult intestine has the ability to agglutinate vegetative cells of[0012]C. botulinum. [S. Arnon, J. Infect. Dis. 154:201 (1986).] Secretory IgA may also act by preventing intestinal bacteria and their products from crossing the cells of the intestine. [S. Arnon, Epidemiol. Rev. 3:45 (1981).] The infant immune system is not primed to do this.

Clinical symptoms of infant botulism range from mild paralysis, to moderate and severe paralysis requiring hospitalization, to fulminant paralysis, leading to sudden death. [S. Arnon, Epidemiol. Rev. 3:45 (1981).][0013]

The chief therapy for severe infant botulism is ventilatory assistance using a mechanical respirator and concurrent elimination of toxin and bacteria using cathartics, enemas, and gastric lavage. There were 68 hospitalizations in California for infant botulism in a single year with a total cost of over $4 million for treatment. [T. L. Frankovich and S. Arnon, West. J. Med. 154:103 (1991).][0014]

Different strains of[0015]Clostridium botulinumeach produce antigenically distinct toxin designated by the letters A-G. Serotype A toxin has been implicated in 26% of the cases of food botulism; types B, E and F have also been implicated in a smaller percentage of the food botulism cases [H. Sugiyama, Microbiol. Rev. 44:419 (1980)]. Wound botulism has been reportedly caused by only types A or B toxins [H. Sugiyama, supra]. Nearly all cases of infant botulism have been caused by bacteria producing either type A or type B toxin. (Exceptionally, one New Mexico case was caused byClostridium botulinumproducing type F toxin and another byClostridium botulinumproducing a type B-type F hybrid.) [S. Arnon, Epidemiol. Rev. 3:45 (1981).] Type C toxin affects waterfowl, cattle, horses and mink. Type D toxin affects cattle, and type E toxin affects both humans and birds.

A trivalent antitoxin derived from horse plasma is commercially available from Connaught Industries Ltd. as a therapy for toxin types A, B, and E. However, the antitoxin has several disadvantages. First, extremely large dosages must be injected intravenously and/or intramuscularly. Second, the antitoxin has serious side effects such as acute anaphylaxis which can lead to death, and serum sickness. Finally, the efficacy of the antitoxin is uncertain and the treatment is costly. [C. O. Tacket et al., Am. J. Med. 76:794 (1984).][0016]

A heptavalent equine botulinal antitoxin which uses only the F(ab′)2 portion of the antibody molecule has been tested by the United States Military. [M. Balady, USAMRDC Newsletter, p. 6 (1991).] This was raised against impure toxoids in those large animals and is not a high titer preparation.[0017]

A pentavalent human antitoxin has been collected from immunized human subjects for use as a treatment for infant botulism. The supply of this antitoxin is limited and cannot be expected to meet the needs of all individuals stricken with botulism disease. In addition, collection of human sera must involve screening out HIV and other potentially serious human pathogens. [P. J. Schwarz and S. S. Arnon, Western J. Med. 156:197 (1992).][0018]

Infant botulism has been implicated as the cause of mortality in some cases of Sudden Infant Death Syndrome (SIDS, also known as crib death). SIDS is officially recognized as infant death that is sudden and unexpected and that remained unexplained despite complete post-mortem examination. The link of SIDS to infant botulism came when fecal or blood specimens taken at autopsy from SIDS infants were found to contain[0019]C. botulinumorganisms and/or toxin in 3-4% of cases analyzed. [D. R. Peterson et al., Rev. Infect. Dis. 1:630 (1979).] In contrast, only 1 of 160 healthy infants (0.6%) hadC. botulinumorganisms in the feces and no botulinal toxin. (S. Arnon et al., Lancet, pp. 1273-76, Jun. 17, 1978.)

In developed countries, SIDS is the number one cause of death in children between one month and one year old. (S. Arnon et al., Lancet, pp. 1273-77, Jun. 17, 1978.) More children die from SIDS in the first year than from any other single cause of death in the first fourteen years of life. In the United States, there are 8,000-10,000 SIDS victims annually. Id.[0020]

What is needed is an effective therapy against infant botulism that is free of dangerous side effects, is available in large supply at a reasonable price, and can be safely and gently delivered so that prophylactic application to infants is feasible.[0021]

Immunization of subjects with toxin preparations has been done in an attempt to induce immunity against botulinal toxins. A[0022]C. botulinumvaccine comprising chemically inactivated (i.e., formaldehyde-treated) type A, B, C, D and E toxin is commercially available for human usage. However, this vaccine preparation has several disadvantages. First, the efficacy of this vaccine is variable (in particular, only 78% of recipients produce protective levels of anti-type B antibodies following administration of the primary series). Second, immunization is painful (deep subcutaneous inoculation is required for administration), with adverse reactions being common (moderate to severe local reactions occur in approximately 6% of recipients upon initial injection; this number rises to approximately 11% of individuals who receive booster injections) [Informational Brochure for the Pentavalent (ABCDE) Botulinum Toxoid, Centers for Disease Control]. Third, preparation of the vaccine is dangerous as active toxin must be handled by laboratory workers.

What is needed are safe and effective vaccine preparations for administration to those at risk of exposure to[0023]C. botulinumtoxins.

[0024]C. difficile

[0025]C. difficile, an organism which gained its name due to difficulties encountered in its isolation, has recently been proven to be an etiologic agent of diarrheal disease. (Sneath et al., p. 1165.).C. difficileis present in the gastrointestinal tract of approximately 3% of healthy adults, and 10-30% of neonates without adverse effect (Swartz, at p. 644); by other estimates,C. difficileis a part of the normal gastrointestinal flora of 2-10% of humans. [G. F. Brooks et al., (eds.) “Infections Caused by Anaerobic Bacteria,” Jawetz, Melnick, &Adelberg's Medical Microbiology,19th ed., pp. 257-262, Appleton & Lange, San Mateo, Calif. (1991).] As these organisms are relatively resistant to most commonly used antimicrobials, when a patient is treated with antibiotics, the other members of the normal gastrointestinal flora are suppressed andC. difficileflourishes, producing cytopathic toxins and enterotoxins. It has been found in 25% of cases of moderate diarrhea resulting from treatment with antibiotics, especially the cephalosporins, clindamycin, and ampicillin. [M. N. Swartz at 644.]

Importantly,[0026]C. difficileis commonly associated with nosocomial infections. The organism is often present in the hospital and nursing home environments and may be carried on the hands and clothing of hospital personnel who care for debilitated and immunocompromised patients. As many of these patients are being treated with antimicrobials or other chemotherapeutic agents, such transmission ofC. difficilerepresents a significant risk factor for disease. (Engelkirk et al., pp. 64-67.)

[0027]C. difficileis associated with a range of diarrhetic illness, ranging from diarrhea alone to marked diarrhea and necrosis of the gastrointestinal mucosa with the accumulation of inflammatory cells and fibrin, which forms a pseudomembrane in the affected area. (Brooks et al.) It has been found in over 95% of pseudomembranous enterocolitis cases. (Swartz, at p. 644.) This occasionally fatal disease is characterized by diarrhea, multiple small colonic plaques, and toxic megacolon. (Swartz, at p. 644.) Although stool cultures are sometimes used for diagnosis, diagnosis is best made by detection of the heat labile toxins present in fecal filtrates from patients with enterocolitis due toC. difficile. (Swartz, at p. 644-645; and Brooks et al., at p. 260.)C. difficiletoxins are cytotoxic for tissue/cell cultures and cause enterocolitis when injected intracecally into hamsters. (Swartz, at p. 644.)

The enterotoxicity of[0028]C. difficileis primarily due to the action of two toxins, designated A and B, each of approximately 300,000 in molecular weight. Both are potent cytotoxins, with toxin A possessing direct enterocytotoxic activity. [Lyerly et al., Infect. Immun. 60:4633 (1992).] Unlike toxin A ofC. perfringens, an organism rarely associated with antimicrobial-associated diarrhea, the toxin ofC. difficileis not a spore coat constituent and is not produced during sporulation. (Swartz, at p. 644.)C. difficiletoxin A causes hemorrhage, fluid accumulation and mucosal damage in rabbit ileal loops and appears to increase the uptake of toxin B by the intestinal mucosa. Toxin B does not cause intestinal fluid accumulation, but it is 1000 times more toxic than toxin A to tissue culture cells and causes membrane damage. Although both toxins induce similar cellular effects such as actin disaggregation, differences in cell specificity occurs.

Both toxins are important in disease. [Borriello et al., Rev. Infect. Dis., 12(suppl. 2):S185 (1990); Lyerly et al., Infect. Immun., 47:349 (1985); and Rolfe, Infect. Immun., 59:1223 (1990).] Toxin A is thought to act first by binding to brush border receptors, destroying the outer mucosal layer, then allowing toxin B to gain access to the underlying tissue. These steps in pathogenesis would indicate that the production of neutralizing antibodies against toxin A may be sufficient in the prophylactic therapy of CDAD. However, antibodies against toxin B may be a necessary additional component for an effective therapeutic against later stage colonic disease. Indeed, it has been reported that animals require antibodies to both toxin A and toxin B to be completely protected against the disease. [Kim and Rolfe, Abstr. Ann. Meet. Am. Soc. Microbiol., 69:62 (1987).][0029]

[0030]C. difficilehas also been reported to produce other toxins such as an enterotoxin different from toxins A and B [Banno et al., Rev. Infect. Dis., 6(Suppl. 1:S11-S20 (1984)], a low molecular weight toxin [Rihn et al., Biochem. Biophys. Res. Comm., 124:690-695 (1984)], a motility altering factor [Justus et al., Gastroenterol., 83:836-843 (1982)], and perhaps other toxins. Regardless,C. difficilegastrointestinal disease is of primary concern.

It is significant that due to its resistance to most commonly used antimicrobials,[0031]C. difficileis associated with antimicrobial therapy with virtually all antimicrobial agents (although most commonly ampicillin, clindamycin and cephalosporins). It is also associated with disease in patients undergoing chemotherapy with such compounds as methotrexate, 5-fluorouracil, cyclophosphamide, and doxorubicin. [S. M. Finegold et al.,Clinical Guide to Anaerobic Infections, pp. 88-89, Star Publishing Co., Belmont, Calif. (1992).]

Treatment of[0032]C. difficiledisease is problematic, given the high resistance of the organism. Oral metronidazole, bacitracin and vancomycin have been reported to be effective. (Finegold et al., p. 89.) However there are problems associated with treatment utilizing these compounds. Vancomycin is very expensive, some patients are unable to take oral medication, and the relapse rate is high (20-25%), although it may not occur for several weeks. Id.

[0033]C. difficiledisease would be prevented or treated by neutralizing the effects of these toxins in the gastrointestinal tract. Thus, what is needed is an effective therapy againstC. difficiletoxin that is free of dangerous side effects, is available in large supply at a reasonable price, and can be safely delivered so that prophylactic application to patients at risk of developing pseudomembranous enterocolitis can be effectively treated.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the reactivity of anti-[0034]C. botulinumIgY by Western blot.

FIG. 2 shows the IgY antibody titer to[0035]C. botulinumtype A toxoid in eggs, measured by ELISA.

FIG. 3 shows the results of[0036]C. difficiletoxin A neutralization assays.

FIG. 4 shows the results of[0037]C. difficiletoxin B neutralization assays.

FIG. 5 shows the results of[0038]C. difficiletoxin B neutralization assays.

FIG. 6 is a restriction map of[0039]C. difficiletoxin A gene, showing sequences of primers 1-4 (SEQ ID NOS:1-4).

FIG. 7 is a Western blot of[0040]C. difficiletoxin A reactive protein.

FIG. 8 shows[0041]C. difficiletoxin A expression constructs.

FIG. 9 shows[0042]C. difficiletoxin A expression constructs.

FIG. 10 shows the purification of recombinant[0043]C. difficiletoxin A.

FIG. 11 shows the results of[0044]C. difficiletoxin A neutralization assays with antibodies reactive to recombinant toxin A.

FIG. 12 shows the results for a[0045]C. difficiletoxin A neutralization plate.

FIG. 13 shows the results for a[0046]C. difficiletoxin A neutralization plate.

FIG. 14 shows the results of recombinant[0047]C. difficiletoxin A neutralization assays.

FIG. 15 shows[0048]C. difficiletoxin A expression constructs.

FIG. 16 shows a chromatograph plotting absorbance at 280 nm against retention time for a pMA1870-680 IgY PEG preparation.[0049]

FIG. 17 shows two recombinant[0050]C. difficiletoxin B expression constructs.

FIG. 18 shows[0051]C. difficiletoxin B expression constructs.

FIG. 19 shows[0052]C. difficiletoxin B expression constructs.

FIG. 20 shows[0053]C. difficiletoxin B expression constructs.

FIG. 21 is an SDS-PAGE gel showing the purification of recombinant[0054]C. difficiletoxin B fusion protein.

FIG. 22 is an SDS-PAGE gel showing the purification of two histidine-tagged recombinant[0055]C. difficiletoxin B proteins.

FIG. 23 shows[0056]C. difficiletoxin B expression constructs.

FIG. 24 is a Western blot of[0057]C. difficiletoxin B reactive protein.

FIG. 25 shows[0058]C. botulinumtype A toxin expression constructs; constructs used to provideC. botulinumorC. difficilesequences are also shown.

FIG. 26 is an SDS-PAGE gel stained with Coomaisse blue showing the purification of recombinant[0059]C. botulinumtype A toxin fusion proteins.

FIG. 27 shows[0060]C. botulinumtype A toxin expression constructs; constructs used to provideC. botulinumsequences are also shown.

FIG. 28 is an SDS-PAGE gel stained with Coomaisse blue showing the purification of pHisBot protein using the Ni-NTA resin.[0061]

FIG. 29 is an SDS-PAGE gel stained with Coomaisse blue showing the expression of pHisBot protein in BL21(DE3) and BL21(DE3)pLysS host cells.[0062]

FIG. 30 is an SDS-PAGE gel stained with Coomaisse blue showing the purification of pHisBot protein using a batch absorption procedure.[0063]

FIG. 31 is an SDS-PAGE gel stained with Coomaisse blue showing the purification of pHisBot and pHisBot(native) proteins using a Ni-NTA column.[0064]

FIG. 32 is an SDS-PAGE gel stained with Coomaisse blue showing the purification of pHisBotA protein expressed in pHisBotA(syn) kan lacIq T7/pACYCGro/BL21(DE3) cells using an IDA column.[0065]

FIG. 33 is an SDS-PAGE gel stained with Coomaisse blue showing the purification of pHisBotA, pHisBotB and pHisBotE proteins by IDA chromatography followed by chromatography on S-100 to remove folding chaperones.[0066]

FIG. 34 is an SDS-PAGE gel stained with Coomaisse blue showing the extracts derived from pHisBotB amp T7lac/BL21(DE3) cells before and after purification on a Ni-NTA column.[0067]

FIG. 35 is an SDS-PAGE gel run under native conditions and stained with Coomaisse blue showing the removal of folding chaperones from IDA-purified BotB protein using a S-100 column.[0068]

FIG. 36 is an SDS-PAGE gel stained with Coomaisse blue showing proteins that eluted during an imidazole step gradient applied to a IDA column containing a lysate of pHisBotB kan laciq T7/pACYCGro/BL21(DE3) cells.[0069]

FIG. 37 is an SDS-PAGE gel run under native conditions and stained with Coomaisse blue showing IDA-purified BotB protein before and after ultrafiltration.[0070]

FIG. 38 is an SDS-PAGE gel stained with Coomaisse blue showing the purification of BotE protein using a NiNTA column.[0071]

FIG. 39 is an SDS-PAGE gel stained with Coomaisse blue showing extracts derived from pHisBotA kan T7 lac/BL21(DE3) pLysS cells grown in fermentation culture.[0072]

FIG. 40 is a chromatograrn showing proteins present after IDA-purified BotE protein was applied to a S-100 column.[0073]

DEFINITIONS

To facilitate understanding of the invention; a number of terms are defined below.[0074]

As used herein, the term “neutralizing” is used in reference to antitoxins, particularly antitoxins comprising antibodies, which have the ability to prevent the pathological actions of the toxin against which the antitoxin is directed.[0075]

As used herein, the term “overproducing” is used in reference to the production of clostridial toxin polypeptides in a host cell and indicates that the host cell is producing more of the clostridial toxin by virtue of the introduction of nucleic acid sequences encoding said clostridial toxin polypeptide than would be expressed by said host cell absent the introduction of said nucleic acid sequences. To allow ease of purification of toxin polypeptides produced in a host cell it is preferred that the host cell express or overproduce said toxin polypeptide at a level greater than 1 mg/liter of host cell culture.[0076]

“A host cell capable of expressing a recombinant protein at a level greater than or equal to 5% of the total cellular protein” is a host cell in which the recombinant protein represents at least 5% of the total cellular protein. To determine what percentage of total cellular protein the recombinant protein represents, the following steps are taken. A total of 10 OD[0077]₆₀₀units of recombinant host cells (e.g., 200 μl of cells at OD₆₀₀=50/ml) are removed (at a timepoint known to represent the peak of expression of the desired recombinant protein) to a 1.5 ml microfuge tube and pelleted for 2 min at maximum rpm in a microfuge. The pellets are resuspended in 1 ml of 50 mM NAHPO₄, 0.5 M NaCl, 40 mM imidazole buffer (pH 6.8) containing 1 mg/ml lysozyme. The samples are incubated for 20 min at room temperature and stored ON at −70° C. Samples are thawed completely at room temperature and sonicated 2×10 seconds with a Branson Sonifier 450 microtip probe at # 3 power setting. The samples are centrifuged for 5 min. at maximum rpm in a microfuge. An aliquot (20 μl) of the protein sample is removed to 20μl 2× sample buffer (this represents the total protein extract). The samples are heated to 95° C. for 5 min, then cooled and 5 or 10 μl are loaded onto 12.5% SDS-PAGE gels. High molecular weight protein markers are also loaded to allow for estimation of the MW of identified recombinant proteins. After electrophoresis, protein is detected generally by staining with Coomassie blue and the stained gel is scanned using a densitometer to determine the percentage of protein present in each band. In this manner, the percentage of protein present in the band corresponding to the recombinant protein of interest may be determined. It is not necessary that Coomassie blue be employed for the detection of protein, a number of fluorescent dyes [e.g., Sypro orange S-6651 (Molecular Probes, Eugene, Oreg.] may be employed and the stained gel scanned using a fluoroimager [e.g., Fluor Imager SI (Molecular Dynamics, Sunnyvale, Calif.)].

“A host cell capable of expressing a recombinant protein as a soluble protein at a level greater than or equal to 0.25% of the total soluble cellular protein” is a host cell in which the amount of soluble recombinant protein present represents at least 0.25% of the total cellular protein. As used herein “total soluble cellular protein” refers to a clarified PEI lysate prepared as described in Example 31(c)(iv). Briefly, cells are harvested following induction of expression of recombinant protein (at a point of maximal expression). The cells are resuspended in cell resuspension buffer (CRB: 50 mM NaPO[0078]₄, 0.5 M NaCl, 40 mM imidazole, pH 6.8) to create a 20% cell suspension (wet weight of cells/volume of CRB) and cell lysates are prepared as described in Example 31(c)(iv) (i.e., sonication or homogenization followed by centrifugation). The cell lysate is then flocculated utilizing polyethyleneimine (PEI) prior to centrifugation. PEI (a 2% solution in dH₂O, pH 7.5 with HCl) is added to the cell lysate to a final concentration of 0.2%, and stirred for 20 min at room temperature prior to centrifugation [8,500 rpm in JA10 rotor (Beckman) for 30 minutes at 4° C.]. This treatment removes RNA, DNA and cell wall components, resulting in a clarified, low viscosity lysate (“PEI clarified lysate”)[The recombinant protein present in the PEI clarified lysate is then purified (e.g., by chromatography on an IDA column for his-tagged proteins). The amount of purified recombinant protein (i.e., the eluted protein) is divided by the concentration of protein present in the PEI clarified lysate (typically 8 mg/ml when using a 20% cell suspension as the starting material) and multiplied by 100 to determine what percentage of total soluble cellular protein is comprised of the soluble recombinant protein (see Example 33b).

As used herein, the term “fusion protein” refers to a chimeric protein containing the protein of interest (ie.,[0079]C. botulinumtoxin A, B, C, D, E, F, or G and fragments thereof) joined to an exogenous protein fragment (the fusion partner which consists of a non-toxin protein). The fusion partner may enhance solubility of theC. botulinumprotein as expressed in a host cell, may provide an affinity tag to allow purification of the recombinant fusion protein from the host cell or culture supernatant, or both. If desired, the fusion protein may be removed from the protein of interest (i.e., toxin protein or fragments thereof) prior to immunization by a variety of enzymatic or chemical means known to the art.

As used herein the term “non-toxin protein” or “non-toxin protein sequence” refers to that portion of a fusion protein which comprises a protein or protein sequence which is not derived from a bacterial toxin protein.[0080]

The term “protein of interest” as used herein refers to the protein whose expression is desired within the fusion protein. In a fusion protein the protein of interest will be joined or fused with another protein or protein domain, the fusion partner, to allow for enhanced stability of the protein of interest and/or ease of purification of the fusion protein.[0081]

As used herein, the term “maltose binding protein” refers to the maltose binding protein of[0082]E. coli. A portion of the maltose binding protein may be added to a protein of interest to generate a fusion protein; a portion of the maltose binding protein may merely enhance the solubility of the resulting fusion protein when expressed in a bacterial host. On the other hand, a portion of the maltose binding protein may allow affinity purification of the fusion protein on an amylose resin.

As used herein, the term “poly-histidine tract” when used in reference to a fusion protein refers to the presence of two to ten histidine residues at either the amino- or carboxy-terminus of a protein of interest. A poly-histidine tract of six to ten residues is preferred. The poly-histidine tract is also defined functionally as being a number of consecutive histidine residues added to the protein of interest which allows the affinity purification of the resulting fusion protein on a nickel-chelate or IDA column.[0083]

As used herein, the term “purified” or “to purify” refers to the removal of contaminants from a sample. For example, antitoxins are purified by removal of contaminating non-immunoglobulin proteins; they are also purified by the removal of immunoglobulin that does not bind toxin. The removal of non-immunoglobulin proteins and/or the removal of immunoglobulins that do not bind toxin results in an increase in the percent of toxin-reactive immunoglobulins in the sample. In another example, recombinant toxin polypeptides are expressed in bacterial host cells and the toxin polypeptides are purified by the removal of host cell proteins; the percent of recombinant toxin polypeptides is thereby increased in the sample. Additionally, the recombinant toxin polypeptides are purified by the removal of host cell components such as lipopolysaccharide (e.g., endotoxin).[0084]

The term “recombinant DNA molecule” as used herein refers to a DNA molecule which is comprised of segments of DNA joined together by means of molecular biological techniques.[0085]

The term “recombinant protein” or “recombinant polypeptide” as used herein refers to a protein molecule which is expressed from a recombinant DNA molecule.[0086]

The term “native protein” as used herein refers to a protein which is isolated from a natural source as opposed to the production of a protein by recombinant means.[0087]

As used herein the term “portion” when in reference to a protein (as in “a portion of a given protein”) refers to fragments of that protein. The fragments may range in size from four amino acid residues to the entire amino acid sequence minus one amino acid.[0088]

As used herein “soluble” when in reference to a protein produced by recombinant DNA technology in a host cell is a protein which exists in solution in the cytoplasm of the host cell; if the protein contains a signal sequence the soluble protein is exported to the periplasmic space in bacteria hosts and is secreted into the culture medium in eucaryotic cells capable of secretion or by bacterial host possessing the appropriate genes (i.e., the kil gene). In contrast, an insoluble protein is one which exists in denatured form inside cytoplasmic granules (called inclusion bodies) in the host cell. High level expression (i.e., greater than 10-20 mg recombinant protein/liter of bacterial culture) of recombinant proteins often results in the expressed protein being found in inclusion bodies in the bacterial host cells. A soluble protein is a protein which is not found in an inclusion body inside the host cell or is found both in the cytoplasm and in inclusion bodies and in this case the protein may be present at high or low levels in the cytoplasm.[0089]

A distinction is drawn between a soluble protein (i.e., a protein which when expressed in a host cell is produced in a soluble form) and a “solubilized” protein. An insoluble recombinant protein found inside an inclusion body may be solubilized (i.e., rendered into a soluble form) by treating purified inclusion bodies with denaturants such as guanidine hydrochloride, urea or sodium dodecyl sulfate (SDS). These denaturants must then be removed from the solubilized protein preparation to allow the recovered protein to renature (refold). Not all proteins will refold into an active conformation after solubilization in a denaturant and removal of the denaturant. Many proteins precipitate upon removal of the denaturant. SDS may be used to solubilize inclusion bodies and will maintain the proteins in solution at low concentration. However, dialysis will not always remove all of the SDS (SDS can form micelles which do not dialyze out); therefore, SDS-solubilized inclusion body protein is soluble but not refolded.[0090]

A distinction is drawn between proteins which are soluble (i.e., dissolved) in a solution devoid of significant amounts of ionic detergents (e.g., SDS) or denaturants (e.g., urea, guanidine hydrochloride) and proteins which exist as a suspension of insoluble protein molecules dispersed within the solution. A soluble protein will not be removed from a solution containing the protein by centrifugation using conditions sufficient to remove bacteria present in a liquid medium (i.e., centrifugation at 12,000×g for 4-5 minutes). For example, to test whether two proteins, protein A and protein B, are soluble in solution, the two proteins are placed into a solution selected from the group consisting of PBS-NaCl (PBS containing 0.5 M NaCl), PBS-NaCl containing 0.2[0091]% Tween 20, PBS, PBS containing 0.2% Tween 20, PBS-C (PBS containing 2 mM CaCl₂), PBS-C containing either 0.1 or 0.5% Tween 20, PBS-C containing either 0.1 or 0.5% NP-40, PBS-C containing either 0.1 or 0.5% Triton X-100, PBS-C containing 0.1% sodium deoxycholate. The mixture containing proteins A and B is then centrifuged at 5000×g for 5 minutes. The supernatant and pellet formed by centrifugation are then assayed for the presence of protein A and B. If protein A is found in the supernatant and not in the pellet [except for minor amounts (i.e., less than 10%) as a result of trapping], protein is said to be soluble in the solution tested. If the majority of protein B is found in the pellet (i.e., greater than 90%), then protein B is said to exist as a suspension in the solution tested.

As used herein, the term “therapeutic amount” refers to that amount of antitoxin required to neutralize the pathologic effects of one or more clostridial toxins in a subject.[0092]

The term “pyrogen” as used herein refers to a fever-producing substance. Pyrogens may be endogenous to the host (e.g., prostaglandins) or may be exogenous compounds (e.g., bacterial endo- and exotoxins, nonbacterial compounds such as antigens and certain steroid compounds, etc.). The presence of pyrogen in a pharmaceutical solution may be detected using the U.S. Pharmacopeia (USP) rabbit fever test (United States Pharmacopeia, Vol. XXII (1990) United States Pharmacopeial Convention, Rockville, Md., p. 151).[0093]

The term “endotoxin” as used herein refers to the high molecular weight complexes associated with the outer membrane of gram-negative bacteria Unpurified endotoxin contains lipids, proteins and carbohydrates. Highly purified endotoxin does not contain protein and is referred to as lipopolysaccharide (LPS). Because unpurified endotoxin is of concern in the production of pharmaceutical compounds (e.g., proteins produced in[0094]E. coliusing recombinant DNA technology), the term endotoxin as used herein refers to unpurified endotoxin. Bacterial endotoxin is a well known pyrogen.

As used herein, the term “endotoxin-free” when used in reference to a composition to be administered parenterally (with the exception of intrathecal administration) to a host means that the dose to be delivered contains less than 5 EU/kg body weight [FDA Guidelines for Parenteral Drugs (December 1987)]. Assuming a weight of 70 kg for an adult human, the dose must contain less than 350 EU to meet FDA Guidelines for parenteral administration. Endotoxin levels are measured herein using the Limulus Amebocyte Lysate (LAL) test (Limulus Amebocyte Lysate Pyrochrome™, Associates of Cape Cod, Inc. Woods Hole, Mass.). To measure endotoxin levels in preparations of recombinant proteins, 0.5 ml of a solution comprising 0.5 mg of purified recombinant protein in 50 mM NaPO[0095]₄, pH 7.0, 0.3M NaCl and 10% glycerol is used in the LAL assay according to the manufacturer's instructions for the endpoint chromogenic without diazo-coupling method [the specific components of the buffer containing recombinant protein to be analyzed in the LAL test are not important; any buffer having a neutral pH may be employed (see for example, alternative buffers employed in Examples 34, 40 and 45)]. Compositions containing less than or equal to than 250 endotoxin units (EU)/mg of purified recombinant protein are herein defined as “substantially endotoxin-free.” Preferably the composition contains less than or equal to 100, and most preferably less than or equal to 60, (EU)/mg of purified recombinant protein. Typically, administration of bacterial toxins or toxoids to adult humans for the purpose of vaccination involves doses of about 10-500 μg protein/dose. Therefore, administration of 10-500 μg of a purified recombinant protein to a 70 kg human, wherein said purified recombinant protein preparation contains 60 EU/mg protein, results in the introduction of only 0.6 to 30 EU (i.e., 0.2 to 8.6% of the maximum allowable endotoxin burden per parenteral dose). Administration of 10-500 μg of a purified recombinant protein to a 70 kg human, wherein said purified recombinant protein preparation contains 250 EU/mg protein, results in the introduction of only 2.5 to 125 EU (ie., 0.7 to 36% of the maximum allowable endotoxin burden per parenteral dose).

The LAL test is accepted by the U.S. FDA as a means of detecting bacterial endotoxins (21 C.F.R. §§ 660.100-105). Studies have shown that the LAL test is equivalent or superior to the USP rabbit pyrogen test for the detection of endotoxin and thus the LAL test can be used as a surrogate for pyrogenicity studies in animals [F. C. Perason,[0096]Pyrogens: endotoxins, LAL testing and depyrogenation, Marcel Dekker, New York (1985), pp.150-155]. The FDA Bureau of Biologics accepts the LAL assay in place of the USP rabbit pyrogen test so long as the LAL assay utilized is shown to be as sensitive as, or more sensitive as the rabbit test [Fed. Reg., 38, 26130 (1980)].

The term “monovalent” when used in reference to a clostridial vaccine refers to a vaccine which is capable of provoking an immune response in a host animal directed against a single type of clostridial toxin. For example, if immunization of a host with[0097]C. botulinumtype A toxin vaccine induces antibodies in the immunized host which protect against a challenge with type A toxin but not against challenge with type B, C, D, E, F or G toxins, then the type A vaccine is said to be monovalent. In contrast, a “multivalent” vaccine provokes an immune response in a host animal directed against several (i.e., more than one) clostridial toxins. For example, if immunization of a host with a vaccine comprisingC. botulinumtype A and B toxins induces the production of antibodies which protect the host against a challenge with both type A and B toxin, the vaccine is said to be multivalent (in particular, this hypothetical vaccine is bivalent).

As used herein the term “immunogenically-effective amount” refers to that amount of an immunogen required to invoke the production of protective levels of antibodies in a host upon vaccination.[0098]

The term “protective level”, when used in reference to the level of antibodies induced upon immunization of the host with an immunogen which comprises a bacterial toxin, means a level of circulating antibodies sufficient to protect the host from challenge with a lethal dose of the toxin.[0099]

As used herein the terms “protein” and “polypeptide” refer to compounds comprising amino acids joined via peptide bonds and are used interchangeably.[0100]

The terms “toxin” and “neurotoxin” when used in reference to toxins produced by members (i.e., species and strains) of the genus Clostridium are used interchangeably and refer to the proteins which are poisonous to nerve tissue.[0101]

Fusion proteins comprising the receptor binding domain (i.e., C fragment) of botulinal toxins may include amino acid residues located beyond the termini of the domains defined above. For example, the pHisBotB protein contains amino acid residues 846-1291 of SEQ ID NO:40; this fusion protein thus comprises the receptor-binding domain for[0103]C. botulinumtype B toxin as defined above (i.e., Ile-848 through Glu-1291). Similarly, pHisBotE contains amino acid residues 827-1252 of SEQ ID NO:50 and pHisBotG contains amino acid residues 851-1297 of SEQ ID NO:77. Thus, both pHisBotE and pHisBotG fusion proteins contain a few amino acids located beyond the N-terminus of the defined receptor-binding domain.

The terms “native gene” or “native gene sequences” are used to indicate DNA sequences encoding a particular gene which contain the same DNA sequences as found in the gene as isolated from nature. In contrast, “synthetic gene sequences” are DNA sequences which are used to replace the naturally occurring DNA sequences when the naturally occurring sequences cause expression problems in a given host cell. For example, naturally-occurring DNA sequences encoding codons which are rarely used in a host cell may be replaced (e.g., by site-directed mutagenesis) such that the synthetic DNA sequence represents a more frequently used codon. The native DNA sequence and the synthetic DNA sequence will preferably encode the same amino acid sequence.[0104]

SUMMARY OF THE INVENTION

The present invention relates to the production of polypeptides derived from toxins particularly in recombinant host cells. In one embodiment, the present invention provides a host cell containing a recombinant expression vector, said vector encoding a protein comprising at least a portion of a[0105]Clostridium botulinumtoxin, said toxin selected from the group consisting of type B toxin and type E toxin. The present invention is not limited by the nature of sequences encoding portions of theC. botulinumtoxin. These sequences may be derived from the native gene sequences or alternatively they may comprise synthetic gene sequences. Synthetic gene sequences are employed when expression of the native gene sequences is problematic in a given host cell (e.g., when the native gene sequences contain sequences resembling yeast transcription termination signals and the desired host cell is a yeast cell).

In one embodiment, the host cell is capable of expressing the recombinant[0106]C. botulinumtoxin protein at a level greater than or equal to 2% to 40% of the total cellular protein and preferably at a level greater than or equal to 5% of the total cellular protein. In another embodiment, the host cell is capable of expressing the recombinantC. botulinumtoxin protein as a soluble protein at a level greater than or equal to 0.25% of the total cellular protein and preferably at a level greater than or equal to 0.25% to 10% of the total cellular protein.

The present invention is not limited by the nature of the host cell employed for the production of recombinant[0107]C. botulinumtoxin proteins. In a preferred embodiment, the host cell is anE. colicell. In another preferred embodiment, the host cell is an insect cell; particularly preferred insect host cells areSpodoptera frugiperda(Sf9) cells. In another preferred embodiment, the host cell is a yeast cell; particularly preferred yeast cells arePichia pastoriscells.

In another embodiment, the invention provides a host cell containing a recombinant expression vector, said vector encoding a fusion protein comprising a non-toxin protein sequence and at least a portion of a[0108]Clostridium botulinumtoxin, said toxin selected from the group consisting of type B toxin and type E toxin. The invention is not limited by the nature of the portion of theClostridium botulinumtoxin selected. In a preferred embodiment, the portion of the toxin comprises the receptor binding domain (i.e., the C fragment of the toxin). The present invention is not limited by the nature of the non-toxin protein sequence employed. In a preferred embodiment, the non-toxin protein sequence comprises a poly-histidine tract. A number of alternative fusion tags or fusion partners are known to the art (e.g., MBP, GST, protein A, etc.) and may be employed for the production of fusion proteins comprising a portion of a botulinal toxin.

The present invention further provides a vaccine comprising a fusion protein, said fusion protein comprising a non-toxin protein sequence and at least a portion of a[0109]Clostridium botulinumtoxin, said toxin selected from the group consisting of type B toxin and type E toxin. The vaccine may be a monovalent vaccine (i.e., containing only a toxin B fusion protein or a toxin E fusion protein), a bivalent vaccine (i.e., containing both a toxin B fusion protein and a toxin E fusion protein) or a trivalent or higher valency vaccine. In a preferred embodiment, the toxin B fusion protein and/or toxin E fusion protein is combined with a fusion protein comprising a non-toxin protein sequence and at least a portion ofClostridium botulinumtype A toxin. The present invention is not limited by the nature of the portion of theClostridium botulinumtoxin selected. In a preferred embodiment, the portion of the toxin comprises the receptor binding domain (i.e., the C fragment of the toxin). The present invention is not limited by the nature of the non-toxin protein sequence employed. In a preferred embodiment, the non-toxin protein sequence comprises a poly-histidine tract. A number of alternative fusion tags or fusion partners are known to the art (e.g., MBP, GST, protein A, etc.) and may be employed for the generation of fusion proteins comprising vaccines. When a fusion partner (i.e., the non-toxin protein sequence) is employed for the production of a recombinantC. botulinaltoxin protein, the fusion partner may be removed from the recombinantC. botulinaltoxin protein if desired (i.e., prior to administration of the protein to a subject) using a variety of methods known to the art (e.g., digestion of fusion proteins containing Factor Xa or thrombin recognition sites with the appropriate enzyme). A number of the pETHis vectors employed herein provide an N-terminal his-tag followed by a Factor Xa cleavage site (see Example 28a); the botulinal C fragment sequences follow the Factor Xa site and thus, Factor Xa can be used to remove the his-tag from the botulinal fusion protein. In a preferred embodiment, the vaccine is substantially endotoxin-free.

The present invention is not limited by the method employed for the generation of vaccine comprising fusion proteins comprising a non-toxin protein sequence and at least a portion of a[0110]Clostridium botulinumtoxin. The fusion proteins may be produced by recombinant DNA means using either native or synthetic gene sequences expressed in a host cell. The present invention is not limited to the production of vaccines using recombinant host cells; cell free in vitro transcription/translation systems may be employed for the expression of the nucleic acid constructs encoding the fusion proteins of the present invention. An example of such a cell-free system is the commercially available TnT™ Coupled Reticulocyte Lysate System (Promega Corporation, Madison, Wis.). Alternatively, the fusion proteins of the present invention may be generated by synthetic means (i.e., peptide synthesis).

The present invention further provides a method of generating antibody directed against a[0111]Clostridium botulinumtoxin comprising: a) providing in any order: i) an antigen comprising a fusion protein comprising a non-toxin protein sequence and at least a portion of aClostridium botulinumtoxin, said toxin selected from the group consisting of type B toxin and type E toxin, and ii) a host; and b) immunizing the host with the antigen so as to generate an antibody. In a preferred embodiment, the antigen used to immunize the host also contains a fusion protein comprising a non-toxin protein sequence and at least a portion ofClostridium botulinumtype A toxin. The present invention is not limited by the nature of the portion of theClostridium botulinumtoxin selected. In a preferred embodiment, the portion of the toxin comprises the receptor binding domain (i.e., the C fragment of the toxin). The present invention is not limited by the nature of the non-toxin protein sequence employed. In a preferred embodiment, the non-toxin protein sequence comprises a poly-histidine tract. A number of alternative fusion tags or fusion partners are known to the art (e.g., MBP, GST, protein A, etc.) and may be employed for the generation of fusion proteins comprising vaccines. When a fusion partner (i.e., the non-toxin protein sequence) is employed for the production of a recombinantC. botulinaltoxin protein, the fusion partner may be removed from the recombinantC. botulinaltoxin protein if desired (i.e., prior to administration of the protein to a subject) using a variety of methods known to the art (e.g., digestion of fusion proteins containing Factor Xa or thrombin recognition sites with the appropriate enzyme).

The present invention is not limited by the nature of the host employed for the production of the antibodies of the invention. In a preferred embodiment, the host is a mammal, preferably a human. The antibodies of the present invention may be generated using non-mammalian hosts such as birds, preferably chickens. In a preferred embodiment the method of the present invention further comprised the step c) of collecting the antibodies from the host. In yet another embodiment, the method of the present invention further comprises the step d) of purifying the antibodies.[0112]

The present invention further provides antibodies raised according to the above methods.[0113]

The present invention further contemplates multivalent vaccines comprising at least two recombinant[0114]C. botulinumtoxin proteins derived from the group consisting ofC. botulinumserotypes A, B, C, D, E, F, and G. The invention contemplates bivalent, trivalent, quadravalent, pentavalent, heptavalent and septivalent vaccines comprising recombinantC. botulinumtoxin proteins. Preferably the recombinantC. botulinumtoxin protein comprises the receptor binding domain (i.e., C fragment) of the toxin.

DESCRIPTION OF THE INVENTION

The present invention contemplates vaccinating humans and other animals with polypeptides derived from[0115]C. botulinumneurotoxins which are substantially endotoxin-free. These botulinal peptides are also useful for the production of antitoxin. Anti-botulinal toxin antitoxin is useful for the treatment of patients effected by or at risk of symptoms due to the action ofC. botulinumtoxins. The organisms, toxins and individual steps of the present invention are described separately below.

I. Clostridium Species, Clostridial Diseases and Associated Toxins[0116]

A preferred embodiment of the method of the present invention is directed toward obtaining antibodies against Clostridium species, their toxins, enzymes or other metabolic by-products, cell wall components, or synthetic or recombinant versions of any of these compounds. It is contemplated that these antibodies will be produced by immunization of humans or other animals. It is not intended that the present invention be limited to any particular toxin or any species of organism. In one embodiment, toxins from all Clostridium species are contemplated as immunogens. Examples of these toxins include the neuraminidase toxin of[0117]C. butyricum, C. sordelliitoxins HT and LT, toxins A, B, C, D, E, F, and G ofC. botulinumand the numerous C perfringens toxins. In one preferred embodiment, toxins A, B, and E ofC. botulinumare contemplated as immunogens. Table 2 above lists various Clostridium species, their toxins and some antigens associated with disease.

TABLE 2


Clostridial Toxins

Organism	Toxins and Disease-Associated Antigens

C. botulinum	A, B, C₁, C₂, D, E, F, G
C. butyricum	Neuraminidase
C. difficile	A, B, Enterotoxin (not A nor B), Motility
	Altering Factor, Low Molecular Weight Toxin, Others
C. perfringens	α, β, ε, ι, γ, δ, ν, θ, κ, λ, μ, υ
C. sordelli/	HT, LT, α, β, γ
C. bifermentans
C. novyi	α, β, γ, δ, ε, ζ, ν, θ
C. septicum	α, β, γ, δ
C. histolyticum	α, β, γ, δ, ε plus additional enzymes
C. chauvoei	α, β, γ, δ

It is not intended that antibodies produced against one toxin will only be used against that toxin. It is contemplated that antibodies directed against one toxin (e.g.,[0118]C. perfringenstype A enterotoxin) may be used as an effective therapeutic against one or more toxin(s) produced by other members of the genus Clostridium or other toxin producing organisms (e.g.,Bacillus cereus, Staphylococcus aureus, Streptococcus mutans, Acinetobacter calcoaceticus, Pseudomonas aeruginosa, other Pseudomonas species, etc.). It is further contemplated that antibodies directed against the portion of the toxin which binds to mammalian membranes (e.g.,C. perfringensenterotoxin A) can also be used against other organisms. It is contemplated that these membrane binding domains are produced synthetically and used as immunogens.

II. Obtaining Antibodies in Non-Mammals[0119]

A preferred embodiment of the method of the present invention for obtaining antibodies involves immunization. However, it is also contemplated that antibodies could be obtained from non-mammals without immunization. In the case where no immunization is contemplated, the present invention may use non-mammals with preexisting antibodies to toxins as well as non-mammals that have antibodies to whole organisms by virtue of reactions with the administered antigen. An example of the latter involves immunization with synthetic peptides or recombinant proteins sharing epitopes with whole organism components.[0120]

In a preferred embodiment, the method of the present invention contemplates immunizing non-mammals with bacterial toxin(s). It is not intended that the present invention be limited to any particular toxin. In one embodiment, toxin from all clostridial bacteria sources (see Table 2) are contemplated as immunogens. Examples of these toxins are[0121]C. butyricumneuraminidase toxin, toxins A, B, C, D, E, F, and G fromC. botulinum, C. perfringenstoxins α, β, ε, and τ, andC. sordelliitoxins HT and LT. In a preferred embodiment,C. botulinumtoxins A, B, C, D, E, and F (or fragments thereof) are contemplated as immunogens.

When immunization is used, the preferred non-mammal is from the class Aves. All birds are contemplated (e.g., duck, ostrich, emu, turkey, etc.). A preferred bird is a chicken. Importantly, chicken antibody does not fix marnmalian complement. [See H. N. Benson et al., J. Immunol. 87:616 (1961).] Thus, chicken antibody will normally not cause a complement-dependent reaction. [A. A. Benedict and K. Yamaga, “[0123]Immunoglobulins and Antibody Production in Avian Species,” in Comparative Immunology(J. J. Marchaloni, ed.), pp. 335-375, Blackwell, Oxford (1966).] Thus, the preferred antitoxins of the present invention will not exhibit complement-related side effects observed with antitoxins known presently.

When birds are used, it is contemplated that the antibody will be obtained from either the bird serum or the egg. A preferred embodiment involves collection of the antibody from the egg. Laying hens transport immunoglobulin to the egg yolk (“IgY”) in concentrations equal to or exceeding that found in serum. [See R. Patterson et al., J. Immunol. 89:272 (1962); and S. B. Carroll and B. D. Stollar, J. Biol. Chem. 258:24 (1983).] In addition, the large volume of egg yolk produced vastly exceeds the volume of serum that can be safely obtained from the bird over any given time period. Finally, the antibody from eggs is purer and more homogeneous; there is far less non-immunoglobulin protein (as compared to serum) and only one class of immunoglobulin is transported to the yolk.[0124]

When considering immunization with toxins, one may consider modification of the toxins to reduce the toxicity. In this regard, it is not intended that the present invention be limited by immunization with modified toxin. Unmodified (“native”) toxin is also contemplated as an immunogen.[0125]

It is also not intended that the present invention be limited by the type of modification—if modification is used. The present invention contemplates all types of toxin modification, including chemical and heat treatment of the toxin. The preferred modification, however, is formaldehyde treatment.[0126]

It is not intended that the present invention be limited to a particular mode of immunization; the present invention contemplates all modes of immunization, including subcutaneous, intramuscular, intraperitoneal, and intravenous or intravascular injection, as well as per os administration of immunogen.[0127]

The present invention further contemplates immunization with or without adjuvant. (Adjuvant is defined as a substance known to increase the immune response to other antigens when administered with other antigens.) If adjuvant is used, it is not intended that the present invention be limited to any particular type of adjuvant—or that the same adjuvant, once used, be used all the time. While the present invention contemplates all types of adjuvant, whether used separately or in combinations, the preferred use of adjuvant is the use of Complete Freund's Adjuvant followed sometime later with Incomplete Freund's Adjuvant. Another preferred use of adjuvant is the use of Gerbu Adjuvant. The invention also contemplates the use of RIBI fowl adjuvant and Quil A adjuvant.[0128]

When immunization is used, the present invention contemplates a wide variety of immunization schedules. In one embodiment, a chicken is administered toxin(s) on day zero and subsequently receives toxin(s) in intervals thereafter. It is not intended that the present invention be limited by the particular intervals or doses. Similarly, it is not intended that the present invention be limited to any particular schedule for collecting antibody. The preferred collection time is sometime after[0129]day 100.

Where birds are used and collection of antibody is performed by collecting eggs, the eggs may be stored prior to processing for antibody. It is preferred that eggs be stored at 4° C. for less than one year.[0130]

It is contemplated that chicken antibody produced in this mariner can be buffer-extracted and used analytically. While unpurified, this preparation can serve as a reference for activity of the antibody prior to further manipulations (e.g., immunoaffinity purification).[0131]

III. Increasing the Effectiveness of Antibodies[0132]

When purification is used, the present invention contemplates purifying to increase the effectiveness of both non-mammalian antitoxins and mammalian antitoxins. Specifically, the present invention contemplates increasing the percent of toxin-reactive immunoglobulin. The preferred purification approach for avian antibody is polyethylene glycol (PEG) separation.[0133]

The present invention contemplates that avian antibody be initially purified using simple, inexpensive procedures. In one embodiment, chicken antibody from eggs is purified by extraction and precipitation with PEG. PEG purification exploits the differential solubility of lipids (which are abundant in egg yolks) and yolk proteins in high concentrations of PEG 8000. [Polson et al., Immunol. Comm. 9:495 (1980).] The technique is rapid, simple, and relatively inexpensive and yields an immunoglobulin fraction that is significantly purer in terms of contaminating non-immunoglobulin proteins than the comparable ammonium sulfate fractions of mammalian sera and horse antibodies. The majority of the PEG is removed from the precipitated chicken immunoglobulin by treatment with ethanol. Indeed, PEG-purified antibody is sufficiently pure that the present invention contemplates the use of PEG-purified antitoxins in the passive immunization of intoxicated humans and animals.[0134]

IV. Treatment[0135]

The present invention contemplates antitoxin therapy for humans and other animals intoxicated by bacterial toxins. A preferred method of treatment is by intravenous administration of anti-boutlinal antitoxin; oral administration is also contemplated for other clostridial antitoxins.[0136]

A. Dosage of Antitoxin[0137]

It was noted by way of background that a balance must be struck when administering currently available antitoxin which is usually produced in large animals such as horses; sufficient antitoxin must be administered to neutralize the toxin, but not so much antitoxin as to increase the risk of untoward side effects. These side effects are caused by: i) patient sensitivity to foreign (e.g, horse) proteins; ii) anaphylactic or immunogenic properties of non-immunoglobulin proteins; iii) the complement fixing properties of mammalian antibodies; and/or iv) the overall burden of foreign protein administered. It is extremely difficult to strike this balance when, as noted above, the degree of intoxication (and hence the level of antitoxin therapy needed) can only be approximated.[0138]

The present invention contemplates significantly reducing side effects so that this balance is more easily achieved. Treatment according to the present invention contemplates reducing side effects by using PEG-purified antitoxin from birds.[0139]

In one embodiment, the treatment of the present invention contemplates the use of PEG-purified antitoxin from birds. The use of yolk-derived, PEG-purified antibody as antitoxin allows for the administration of: 1) non(mammalian)-complement-fixing, avian antibody; 2) a less heterogeneous mixture of non-immunoglobulin proteins; and 3) less total protein to deliver the equivalent weight of active antibody present in currently available antitoxins. The non-mammalian source of the antitoxin makes it useful for treating patients who are sensitive to horse or other mammalian sera.[0140]

B. Delivery of Antitoxin[0141]

Although it is not intended to limit the route of delivery, the present invention contemplates a method for antitoxin treatment of bacterial intoxication in which delivery of antitoxin is oral. In one embodiment, antitoxin is delivered in a solid form (e.g., tablets). In an alternative embodiment antitoxin is delivered in an aqueous solution. When an aqueous solution is used, the solution has sufficient ionic strength to solubilize antibody protein, yet is made palatable for oral administration. The delivery solution may also be buffered (e.g., carbonate buffer pH 9.5) which can neutralize stomach acids and stabilize the antibodies when the antibodies are administered orally. In one embodiment the delivery solution is an aqueous solution. In another embodiment the delivery solution is a nutritional formula. Preferably, the delivery solution is infant formula. Yet another embodiment contemplates the delivery of lyophiliZed antibody encapsulated or microencapsulated inside acid-resistant compounds.[0142]

Methods of applying enteric coatings to pharmaceutical compounds are well known to the art [companies specializing in the coating of pharmaceutical compounds are available; for example, The Coating Place (Verona, Wis.) and AAI (Wilmington, N.C.)]. Enteric coatings which are resistant to gastric fluid and whose release (i.e., dissolution of the coating to release the pharmaceutical compound) is pH dependent are commercially available [for example, the polymethacrylates Eudragit® L and Eudragit® S (Röhm GmbH)]. Eudragit® S is soluble in intestinal fluid from pH 7.0; this coating can be used to microencapsulate lyophilized antitoxin antibodies and the particles are suspended in a solution having a pH above or below pH 7.0 for oral administration. The microparticles will remain intact and undissolved until they reached the intestines where the intestinal pH would cause them to dissolve thereby releasing the antitoxin.[0143]

The invention contemplates a method of treatment which can be administered for treatment of acute intoxication. In one embodiment, antitoxin is administered orally in either a delivery solution or in tablet form, in therapeutic dosage, to a subject intoxicated by the bacterial toxin which served as immunogen for the antitoxin.[0144]

The invention also contemplates a method of treatment which can be administered prophylactically. In one embodiment, antitoxin is administered orally, in a delivery solution, in therapeutic dosage, to a subject, to prevent intoxication of the subject by the bacterial toxin which served as immunogen for the production of antitoxin. In another embodiment, antitoxin is administered orally in solid form such as tablets or as microencapsulated particles. Microencapsulation of lyophilized antibody using compounds such as Eudragit® (Rohm GmbH) or polyethylene glycol, which dissolve at a wide range of pH units, allows the oral administration of solid antitoxin in a liquid form (i.e., a suspension) to recipients unable to tolerate administration of tablets (e.g., children or patients on feeding tubes). In one preferred embodiment the subject is a child. In another embodiment, antibody raised against whole bacterial organism is administered orally to a subject, in a delivery solution, in therapeutic dosage.[0145]

V. Vaccines Against Clostridial Species[0146]

The invention contemplates the generation of mono- and multivalent vaccines for the protection of an animal (particularly humans) against several clostridial species. Of particular interest are vaccines which stimulate the production of a humoral immune response to[0147]C. botulinum, C. tetaniandC. difficilein humans. The antigens comprising the vaccine preparation may be native or recombinantly produced toxin proteins from the clostridial species listed above. When toxin proteins are used as immunogens they are generally modified to reduce the toxicity. This modification may be by chemical or genetic (i.e., recombinant DNA technology) means. In general genetic detoxification (i.e., the expression of nontoxic fragments in a host cell) is preferred as the expression of nontoxic fragments in a host cell precludes the presence of intact, active toxin in the final preparation. However, when chemical modification is desired, the preferred toxin modification is formaldehyde treatment.

The invention contemplates that recombinant[0148]C. botulinumtoxin proteins be used as antigens in mono- and multivalent vaccine preparations. Soluble, substantially endotoxin-free recombinantC. botulinumtoxin proteins derived from serotypes A, B and E may be used individually (i.e., as mono-valent vaccines) or in combination (i.e., as a multi-valent vaccine). In addition, the recombinantC. botulinumtoxin proteins derived from serotpes A, B and E may be used in conjunction with either recombinant or native toxins or toxoids from other serotypes ofC. botulinum, C. difficileandC. tetanias antigens for the preparation of these mono- and multivalent vaccines. It is contemplated that, due to the structural similarity ofC. botulinumandC. tetanitoxin proteins, a vaccine comprisingC. difficileandbotulinumtoxin proteins (native or recombinant or a mixture thereof) be used to stimulate an immune response againstC. botulinum, C. tetaniandC. difficile.

The present invention further contemplates multi-valent vaccines comprising two or more botulinal toxin proteins selected from the group comprising recombinant[0149]C. botulinumtoxin proteins derived from serotypes A, B, C (including C1 and C2), D, E, F and G.

The adverse consequences of exposure to botulinal toxin would be avoided by immunization of subjects at risk of exposure to the toxin with nontoxic preparations which confer immunity such as chemically or genetically detoxified toxin.[0150]

Vaccines which confer immunity against one or more of the toxin types A, B, E, F and G would be useful as a means of protecting humans from the deleterious effects of those[0151]C. botulinumtoxins known to affect man. Indeed as the possibility exists that humans could be exposed to any of the seven serotypes ofC. botulinumtoxin (e.g., during biological warfare or the production of toxin in a laboratory setting), multivalent vaccines capable of conferring immunity against toxin types A-G (including both C1 and C2 toxins) would be useful for the protection of humans. Vaccines which confer immunity against one or more of the toxin types C, D and E would be useful for veterinary applications.

The botulinal neurotoxin is synthesized as a single polypeptide chain which is processed into a heavy (H; ˜100 kD) and a light (L; ˜50 kD) chain by cleavage with proteolytic enzymes; these two chains are held together via disulfide bonds in the active toxin (referred to as derivative toxin) [B. R. DasGupta and H. Sugiyama, Biochem. Biophys. Res. Commun. 48:108 (1972); reviewed in B. R. DasGupta, J. Physiol. 84:220 (1990), H. Sugiyama, Microbiol. Rev. 44:419 (1980) and C. L. Hatheway, Clin. Microbiol. Rev. 3:66 (1990)]. The heavy chain of the active toxin is cleaved by trypsin to produce two fragments termed H[0152]_C(also referred to as H₁or C) and H_N(also referred to as H₂or B). The H_Cfragment (˜46 kD) comprises the carboxy end of the H chain. The H_Nfragment (˜49 kD) comprises the animo end and remains attached to the L chain (H_NL). Neither H_Cor H_NL is toxic. H_Ccompetes with whole derivative toxin for binding to synaptosomes and therefore H_Cis said to contain the receptor binding site. The H_Cand H_Nfragments of botulinal toxin are analogous to the fragments C and B of tetanus toxin which are produced by papain cleavage. The C fragment of tetanus toxin has been shown to be responsible for the binding of tetanus toxin to purified gangliosides and neuronal cells [Halpern and Loftus, J. Biol. Chem. 288:11188 (1993)].

Antisera raised against purified preparations of isolated botulinal H and L chains have been shown to protect mice against the lethal effects of the toxin; however, the effectiveness of the two antisera differ with the anti-H sera being more potent (H. Sugiyama, supra). While the different botulinal toxins show structural similarity to one another, the different serotypes are reported to be immunologically distinct (i.e., sera raised against one toxin type does not cross-react to a significant degree with other types). Thus, the generation of multivalent vaccines may require the use of more than one type of toxin.[0153]

[0154]C. botulinumtoxin genes from all seven serotypes have been cloned and sequenced (Minton (1995), supra); in addition, partial amino acid sequence is available for a number ofC. botulinumtoxins isolated from different strains within a given serotype. TheC. botulinumtoxins contain about 1250-1300 amino acid residues. On the DNA level, the overall degree of homology betweenC. botulinumserotypes A, B, C, D and E toxins averages between 50 and 60% identity with a greater degree of homology being found between H chain-encoding regions than between those encoding L chains [Whelan et al. (1992) Appl. Environ. Microbiol. 58:2345]. The degree of identity betweenC. botulinumtoxins on the amino acid level reflects the level of DNA sequence homology. The most divergent area of DNA and amino acid sequence is found within the carboxy-terminal area of the variousC. botulinumH chain genes. This portion of the toxin (i.e., H_Cor the C fragment) plays a major role in cell binding. As toxin from different serotypes is thought to bind to distinct cell receptor molecules, it is not surprising that the toxins diverge significantly over this region.

Within a given serotype, small variations in the primary amino acid sequence of the botulinal toxins isolated from different strains has been reported [Whelan et al. (1992), supra and Minton (1995), supra]. The present invention contemplates fusion proteins comprising portions of[0155]C. botulinumtoxins from serotypes A-G including the variants found among different strains within a given serotype. The present invention provides oligonucleotide primers which may be used to amplify the C fragment or receptor-binding region of the toxin gene from various strains ofC. botulinumserotype A, serotype B, serotype C (C1 and C2), serotype D, serotype E, serotype F and serotype G. A large number of different strains ofC. botulinumserotype A, serotype B, serotype C, serotype D serotype E and serotype F are available from the American Type Culture Collection (ATCC; Rockville, Md.). For example, the ATCC provides the following: Type A strains: 174 (ATCC 3502), 457 (ATCC 17862), and NCTC 7272 (ATCC 19397); Type B strains: 34 (ATCC 439), 62A (ATCC 7948), NCA 213 B (ATCC 7949), 13114 (ATCC 8083), 3137 (ATCC 17780), 1347 (ATCC 17841), 2017 (ATCC 17843), 2217 (ATCC 17844), 2254 (ATCC 17845) and VP 1.731 (ATCC 25765); Type C strains: 2220 (ATCC 17782), 2239 (ATCC 17783), 2223 (ATCC 17784; a type C-β strain; C-β strains produce C2 toxin), 662 (ATCC 17849; a type C-α strain; C-α strains produce mainly C1 toxin and a small amount of C2 toxin), 2021 (ATCC 17850; a type C-α strain) and VPI 3803 (ATCC 25766); Type D strains: ATCC 9633, 2023 (ATCC 17851), and VPI 5995 (ATCC 27517); Type E strains: ATCC 43181, 36208 (ATCC 9564), 2231 (ATCC 17786), 2229 (ATCC 17852), 2279 (ATCC 17854) and 2285 (ATCC 17855) and Type F strains: 202F (ATCC 23387), VPI 4404 (ATCC 25764), VPI 2382 (ATCC 27321) and Langeland (ATCC 35415). Type G strain, 113/30 (NCFB 3012) may be obtained from the National Collection of Food Bacteria (NCFB, AFRC Institute of Food Research, Reading, United Kingdom).

Purification methods have been reported for native toxin types A, B, C, D, E, and F [reviewed in G. Sakaguchi, Pharmac. Ther. 19:165 (1983)]. As the different botulinal toxins are structurally related, the invention contemplates the expression of any of the botulinal toxins (e.g., types A-G) as soluble recombinant fusion proteins.[0156]

In particular, methods for purification of the type A[0157]botulinumneurotoxin have been developed [L. J. Moberg and H. Sugiyama, Appl. Environ. Microbiol. 35:878 (1978)]. Immunization of hens with detoxified purified protein results in the generation of neutralizing antibodies [B. S. Thalley et al., inBotulinum and Tetanus Neurotoxins, B. R. DasGupta, ed., Plenum Press, New York (1993), p. 467].

The currently available[0158]C. botulinumpentavalent vaccine comprising chemically inactivated (i.e., formaldehyde treated) type A, B, C, D and E toxins is not adequate. The efficacy is variable (in particular, only 78% of recipients produce protective levels of anti-type B antibodies following administration of the primary series) and immunization is painful (deep subcutaneous inoculation is required for administration), with adverse reactions being common (moderate to severe local reactions occur in approximately 6% of recipients upon initial injection; this number rises to approximately 11% of individuals who receive booster injections) [Informational Brochure for the Pentavalent (ABCDE) Botulinum Toxoid, Centers for Disease Control]. Preparation of this vaccine is dangerous as active toxin must be handled by laboratory workers.

In general, chemical detoxification of bacterial toxins using agents such as formaldehyde, glutaraldehyde or hydrogen peroxide is not optimal for the generation of vaccines or antitoxins. A delicate balance must be struck between too much and too little chemical modification. If the treatment is insufficient, the vaccine may retain residual toxicity. If the treatment is too excessive, the vaccine may lose potency due to destruction of native immunogenic determinants. Another major limitation of using botulinal toxoids for the generation of antitoxins or vaccines is the high production expense. For the above reasons, the development of methods for the production of nontoxic but immunogenic[0159]C. botulinumtoxin proteins is desirable.

The[0160]C. botulinumandC. tetanustoxin proteins have similar structures [reviewed in E. J. Schantz and E. A. Johnson, Microbiol. Rev. 56:80 (1992)]. The carboxy-terminal 50 kD fragment of the tetanus toxin heavy chain (fragment C) is released by papain cleavage and has been shown to be non-toxic and immunogenic. Recombinant tetanus toxin fragment C has been developed as a candidate vaccine antigen [A. J. Makoff et al., Bio/Technology 7:1043 (1989)]. Mice immunized with recombinant tetanus toxin fragment C were protected from challenge with lethal doses of tetanus toxin. No studies have demonstrated that the recombinant tetanus fragment C protein confers immunity against other botulinal toxins such as theC. botulinumtoxins.

Recombinant tetanus fragment C has been expressed in[0161]E. coli(A. J. Makoff et al., Bio/Technology, supra and Nucleic Acids Res. 17:10191 (1989); J. L. Halpern et al., Infect. Immun. 58:1004 (1990)], yeast [M. A. Romanos et al., Nucleic Acids Res. 19:1461 (1991)] and baculovirus [I. G. Charles et al., Infect. Immun. 59:1627 (1991)]. Synthetic tetanus toxin genes had to be constructed to facilitate expression in yeast (M. A. Romanos et al., supra) andE. coli[A. J. Makoff et al., Nucleic Acids Res., supra], due to the high A-T content of the tetanus toxin gene sequences. High A-T content is a common feature of clostridial genes (M. R. Popoff et al., Infect. Immun. 59:3673 (1991); H. F. LaPenotiere et al., inBotulinum and Tetanus Neurotoxins, B. R. DasGupta, ed., Plenum Press, New York (1993), p. 463] which creates expression difficulties inE. coliand yeast due primarily to altered codon usage frequency and fortuitous polyadenylation sites, respectively.

The C fragment of the[0162]C. botulinumtype A neurotoxin heavy chain has been evaluated as a vaccine candidate. TheC. botulinumtype A neurotoxin gene has been cloned and sequenced [D. E. Thompson et al., Eur. J. Biochem. 189:73 (1990)]. The C fragment of the type A toxin was expressed as either a fusion protein comprising the botulinal C fragment fused with the maltose binding protein (MBP) or as a native protein [H. F. LaPenotiere et al., (1993) supra, H. F. LaPenotiere et al., Toxicon. 33:1383 (1995) and Middlebrook and Brown (1995), Curr. Top. Microbiol. Imrnunol. 195:89-122]. The plasmid construct encoding the native protein was reported to be unstable, while the fusion protein was expressed primarily in inclusion bodies as insoluble protein. Immunization of mice with crudely purified MBP fusion protein resulted in protection against IP challenge with 3 LD₅₀doses of toxin [LaPenotiere et al., (1993) and (1995), supra]. However, this recombinantC. botulinumtype A toxin C fragment/MBP fusion protein is not a suitable immunogen for the production of vaccines as it is expressed as an insoluble protein inE. coli. Furthermore, this recombinantC. botulinumtype A toxin C fragment/MBP fusion protein was not shown to be substantially free of endotoxin contamination. Experience with recombinantC. botulinumtype A toxin C fragment/MBP fusion proteins shows that the presence of the MBP on the fusion protein greatly complicates the removal of endotoxin from preparations of the recombinant fusion protein (see Ex. 24, infra). Expression of a synthetic gene encodingC. botulinumtype A toxin C fragment as a soluble protein excreted from insect cells has been reported [Middlebrook and Brown (1995), supra]; no details regarding the level of expression achieved or the presence of endotoxin or other pyrogens were provided. Like the insoluble protein expressed inE. coli, immunization with the recombinant protein produced in insect cells was reported to protect mice from challenge withC. botulinumtoxin A.

Inclusion body protein must be solubilized prior to purification and/or administration to a host. The harsh treatment of inclusion body protein needed to accomplish this solubilization may reduce the immunogenicity of the purified protein. Ideally, recombinant proteins to be used as vaccines are expressed as soluble proteins at high levels (i.e., greater than or equal to about 0.75% of total cellular protein) in[0163]E. colior other host cells (e.g., yeast, insect cells, etc.). This facilitates the production and isolation of sufficient quantities of the immunogen in a highly purified form (i.e., substantially free of endotoxin or other pyrogen contamination). The ability to express recombinant toxin proteins as soluble proteins inE. coliis advantageous due to the low cost of growth compared to insect or mammalian tissue culture cells.

The[0164]C. botulinumtype B neurotoxin gene has been cloned and sequenced from two strains ofC. botulinumtype B [Whelan et al. (1992) Appl. Environ. Microbiol. 58:2345 (Danish strain) and Hutson et al. (1994) Curr. Microbiol. 28:101 (Eklund 17B strain)]. The nucleotide sequence of the toxin gene derived from the Eklund 17B strain (ATCC 25765) is available from the EMBL/GenBank sequence data banks under the accession number X71343; the nucleotide sequence of the coding region is listed in SEQ ID NO:39. The amino acid sequence of theC. botulinumtype B neurotoxin derived from the strain Eklund 17B is listed in SEQ ID NO:40. The nucleotide sequence of theC. botulinumserotype B toxin gene derived from the Danish strain is listed in SEQ ID NO:41. The amino acid sequence of theC. botulinumtype B neurotoxin derived from the Danish strain is listed in SEQ ID NO:42.

The[0165]C. botulinumtype B neurotoxin gene is synthesized as a single polypeptide chain which is processed to form a dimer composed of a light and a heavy chain linked via disulfide bonds. The light chain is responsible for pharmacological activity (i.e., inhibition of the release of acetylcholine at the neuromuscular junction). The N-terminal portion of the heavy chain is thought to mediate channel formation while the C-terminal portion mediates toxin binding; the type B neurotoxin has been reported to exist as a mixture of predominantly single chain with some double chain (Whelan et al., supra). The 50 kD carboxy-terminal portion of the heavy chain is referred to as the C fragment or the H_Cdomain. The present invention reports for the first time, the expression of the C fragment ofC. botulinumtype B toxin in heterologous hosts (e.g.,E. coli).

The[0166]C. botulinumtype E neurotoxin gene has been cloned and sequenced from a number of different strains [Poulet et al. (1992) Biochem. Biophys. Res. Commun. 183:107; Whelan et al. (1992) Eur. J. Biochem. 204:657; and Fujii et al. (1993) J. Gen. Microbiol. 139:79]. The nucleotide sequence of the type E toxin gene is available from the EMBL sequence data bank under accession numbers X62089 (strain Beluga) and X62683 (strain NCTC 11219); the nucleotide sequence of the coding region (strain Beluga) is listed in SEQ ID NO:45. The amino acid sequence of theC. botulinumtype E neurotoxin derived from strain Beluga is listed in SEQ ID NO:46. The type E neurotoxin gene is synthesized as a single polypeptide chain which may be converted to a double-chain form (i.e., a heavy chain and a light chain) by cleavage with trypsin; unlike the type A neurotoxin, the type E neurotoxin exists essentially only in the single-chain form. The 50 kD carboxy-terminal portion of the heavy chain is referred to as the C fragment or the H_Cdomain. The present invention reports for the first time, the expression of the C fragment ofC. botulinumtype E toxin in heterologous hosts (e.g.,E. coli).

The[0167]C. botulinumtype C1, D, F and G neurotoxin genes have been cloned and sequenced. The nucleotide and amino acid sequences of these genes and toxins are provided herein. The invention provides methods for the expression of the C fragment from each of these toxin genes in heterologous hosts and the purification of the resulting recombinant proteins.

The subject invention provides methods which allow the production of soluble[0168]C. botulinumtoxin proteins in economical host cells (e.g.,E. coli). In addition the subject invention provides methods which allow the production of soluble botulinal toxin proteins in yeast and insect cells. Further, methods for the isolation of purified solubleC. botulinumtoxin proteins which are suitable for immunization of humans and other animals are provided. These soluble, purified preparations ofC. botulinumtoxin proteins provide the basis for improved vaccine preparations and facilitate the production of antitoxin.

When recombinant clostridial toxin proteins produced in gram-negative bacteria (e.g.,[0169]E. coli) are used as vaccines, they are purified to remove endotoxin prior to administration to a host animal. In order to vaccinate a host, an immunogenically-effective amount of purified substantially endotoxin-free recombinant clostridial toxin protein is administered in any of a number of physiologically acceptable carriers known to the art. When administered for the purpose of vaccination, the purified substantially endotoxin-free recombinant clostridial toxin protein may be used alone or in conjunction with known adjutants, including potassium alum, aluminum phosphate, aluminum hydroxide, Gerbu adjuvant (GmDP; C.C. Biotech Corp.), RIBI adjuvant (MPL; RIBI Immunochemical Research, Inc.), QS21 (Carnbridge Biotech). The alum and aluminum-based adjutants are particularly preferred when vaccines are to be administered to humans; however, any adjuvant approved for use in humans may be employed. The route of immunization may be nasal, oral, intramuscular, intraperitoneal or subcutaneous.

The invention contemplates the use of soluble, substantially endotoxin-free preparations of fusion proteins comprising the C fragment of the[0170]C. botulinumtype A, B, C, D, E, F, and G toxin as vaccines. In one embodiment, the vaccine comprises the C fragment of either theC. botulinumtype A, B, C, D, E, F, or G toxin and a poly-histidine tract (also called a histidine tag). In a particularly preferred embodiment, a fusion protein comprising the histidine tagged C fragment is expressed using the pET series of expression vectors (Novagen). The pET expression system utilizes a vector containing the T7 promoter which encodes the fusion protein and a host cell which can be induced to express the T7 DNA polymerase (i.e., a DE3 host strain). The production of C fragment fusion proteins containing a histidine tract is not limited to the use of a particular expression vector and host strain. Several commercially available expression vectors and host strains can be used to express the C fragment protein sequences as a fusion protein containing a histidine tract (For example, the pQE series (pQE-8, 12, 16, 17, 18, 30, 31, 32, 40, 41, 42, 50, 51, 52, 60 and 70) of expression vectors (Qiagen) which are used with the host strains M15[pREP4] (Qiagen) and SG13009[pREP4] (Qiagen) can be used to express fusion proteins containing six histidine residues at the amino-terminus of the fusion protein). Furthermore a number of commercially available expression vectors which provide a histidine tract also provide a protease cleavage site between the histidine tract and the protein of interest (e.g., botulinal toxin sequences). Cleavage of the resulting fusion protein with the appropriate protease will remove the histidine tag from the protein of interest (e.g., botulinal toxin sequences) (see Example 28a, infra). Removal of the histidine tag may be desirable prior to administration of the recombinant botulinal toxin protein to a subject (e.g., a human).

VI. Detection of Toxin[0171]

The invention contemplates detecting bacterial toxin in a sample. The term “sample” in the present specification and claims is used in its broadest sense. On the one hand it is meant to include a specimen or culture. On the other hand, it is meant to include both biological and environmental samples.[0172]

Biological samples may be animal, including human, fluid, solid (e.g., stool) or tissue; liquid and solid food products and ingredients such as dairy items, vegetables, meat and meat by-products, and waste. Environmental samples include environmental material such as surface matter, soil, water and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, disposable and non-disposable items. These examples are not to be construed as limiting the sample types applicable to the present invention.[0173]

The invention contemplates detecting bacterial toxin by a competitive immunoassay method that utilizes recombinant toxin A and toxin B proteins, antibodies raised against recombinant bacterial toxin proteins. A fixed amount of the recombinant toxin proteins are immobilized to a solid support (e.g., a microtiter plate) followed by the addition of a biological sample suspected of containing a bacterial toxin. The biological sample is first mixed with affinity-purified or PEG fractionated antibodies directed against the recombinant toxin protein. A reporter reagent is then added which is capable of detecting the presence of antibody bound to the immobilized toxin protein. The reporter substance may comprise an antibody with binding specificity for the antitoxin attached to a molecule which is used to identify the presence of the reporter substance. If toxin is present in the sample, this toxin will compete with the immobilized recombinant toxin protein for binding to the anti-recombinant antibody thereby reducing the signal obtained following the addition of the reporter reagent. A control is employed where the antibody is not mixed with the sample. This gives the highest (or reference) signal.[0174]

The invention also contemplates detecting bacterial toxin by a “sandwich” immunoassay method that utilizes antibodies directed against recombinant bacterial toxin proteins. Affinity-purified antibodies directed against recombinant bacterial toxin proteins are immobilized to a solid support (e.g., microtiter plates). Biological samples suspected of containing bacterial toxins are then added followed by a washing step to remove substantially all unbound antitoxin. The biological sample is next exposed to the reporter substance, which binds to antitoxin and is then washed free of substantially all unbound reporter substance. The reporter substance may comprise an antibody with binding specificity for the antitoxin attached to a molecule which is used to identify the presence of the reporter substance. Identification of the reporter substance in the biological tissue indicates the presence of the bacterial toxin.[0175]

It is also contemplated that bacterial toxin be detected by pouring liquids (e.g., soups and other fluid foods and feeds including nutritional supplements for humans and other animals) over immobilized antibody which is directed against the bacterial toxin. It is contemplated that the immobilized antibody will be present in or on such supports as cartridges, columns, beads, or any other solid support medium. In one embodiment, following the exposure of the liquid to the immobilized antibody, unbound toxin is substantially removed by washing. The exposure of the liquid is then exposed to a reporter substance which detects the presence of bound toxin. In a preferred embodiment the reporter substance is an enzyme, fluorescent dye, or radioactive compound attached to an antibody which is directed against the toxin (i.e., in a “sandwich” immunoassay). It is also contemplated that the detection system will be developed as necessary (e.g., the addition of enzyme substrate in enzyme systems; observation using fluorescent light for fluorescent dye systems; and quantitation of radioactivity for radioactive systems).[0176]

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.[0177]

In the disclosure which follows, the following abbreviations apply: ° C. (degrees Centigrade); rpm (revolutions per minute); BBS-Tween (borate buffered saline containing Tween); BSA (bovine serum albumin); ELISA (enzyme-linked immunosorbent assay); CFA (complete Freund's adjuvant); IFA (incomplete Freund's adjuvant); IgG (immunoglobulin G); IgY (immunoglobulin Y); IM (intramuscular); IP (intraperitoneal); IV (intravenous or intravascular); SC (subcutaneous); H[0178]₂O (water); HCl (hydrochloric acid); LD₁₀₀(lethal dose for 100% of experimental animals); aa (amino acid); HPLC (high performance liquid chromatography); kD (kilodaltons); gm (grams); μg (micrograms); mg (milligrams); ng (nanograms); μl (microliters); ml (milliliters); mm (millimeters); nm (nanometers); μm (micrometer); M (molar); mM (millimolar); MW (molecular weight); sec (seconds); min(s) (minute/minutes); hr(s) (hour/hours); MgCl, (magnesium chloride); NaC1 (sodium chloride); Na₂CO₃(sodium carbonate); OD₂₈₀(optical density at 280 nm); OD₆₀₀(optical density at 600 nm); PAGE (polyacrylamide gel electrophoresis); PBS [phosphate buffered saline (150 mM NaCl, 10 mM sodium phosphate buffer, pH 7.2)]; PEG (polyethylene glycol); PMSF (phenylmethylsulfonyl fluoride); SDS (sodium dodecyl sulfate); Tris (tris(hydroxymethyl)aminomethane); Ensure® (Ensure®, Ross Laboratories, Columbus Ohio); Enfamil® (Enfamil®, Mead Johnson); w/v (weight to volume); v/v (volume to volume); Amicon (Amicon, Inc., Beverly, Mass.); Amresco (Amresco, Inc., Solon, Ohio); ATCC (American Type Culture Collection, Rockville, Md.); BBL (Baltimore Biologics Laboratory, (a division of Becton Dickinson), Cockeysville, Md.); Becton Dickinson (Becton Dickinson Labware, Lincoln Park, N.J.); BioRad (BioRad, Richmond, Calif.); Biotech (C-C Biotech Corp., Poway, Calif.); Charles River (Charles River Laboratories, Wilmington, Mass.); Cocalico (Cocalico Biologicals Inc., Reamstown, Pa.); CytRx (CytRx Corp., Norcross, Ga.); Falcon (e.g. Baxter Healthcare Corp., McGaw Park, Ill. and Becton Dickinson); FDA (Federal Food and Drug Administration); Fisher Biotech (Fisher Biotech, Springfield, N.J.); GIBCO (Grand Island Biologic Company/BRL, Grand Island, N.Y.); Gibco-BRL (Life Technologies, Inc., Gaithersburg, Md.); Harlan Sprague Dawley (Harlan Sprague Dawley, Inc., Madison, Wis.); Mallinckrodt (a division of Baxter Healthcare Corp., McGaw Park, Ill.); Millipore (Millipore Corp., Marlborough, Mass.); New England Biolabs (New England Biolabs, Inc., Beverly, Mass.); Novagen (Novagen, Inc., Madison, Wis.); Pharmacia (Pharmacia, Inc., Piscataway, N.J.); Qiagen (Qiagen, Chatsworth, Calif.); Sasco (Sasco, Omaha, Nebr.); Showdex (Showa Denko America, Inc., New York, N.Y.); Sigma (Sigma Chemical Co., St. Louis, Mo.); Sterogene (Sterogene, Inc., Arcadia, Calif.); Tech Lab (Tech Lab, Inc., Blacksburg, Va.); and Vaxcell (Vaxcell, Inc., a subsidiary of CytRX Corp., Norcross, Ga.).

When a recombinant protein is described in the specification it is referred to in a short-hand manner by the amino acids in the toxin sequence present in the recombinant protein rounded to the nearest 10. For example, the recombinant protein pMB1850-2360 contains amino acids 1852 through 2362 of the[0179]C. difficiletoxin B protein. The specification gives detailed construction details for all recombinant proteins such that one skilled in the art will know precisely which amino acids are present in a given recombinant protein.

Example 1Production of High-Titer Antibodies toClostridium difficileOrganisms in a Hen

Antibodies to certain pathogenic organisms have been shown to be effective in treating diseases caused by those organisms. It has not been shown whether antibodies can be raised, against[0180]Clostridium difficile, which would be effective in treating infection by this organism. Accordingly,C. difficilewas tested as immunogen for production of hen antibodies.

To determine the best course for raising high-titer egg antibodies against whole[0181]C. difficileorganisms, different immunizing strains and different immunizing concentrations were examined. The example involved (a) preparation of the bacterial immunogen, (b) immunization, (c) purification of anti-bacterial chicken antibodies, and (d) detection of anti-bacterial antibodies in the purified IgY preparations.

a) Preparation of Bacterial Immunogen[0182]

[0183]C. difficilestrains 43594 (serogroup A) and 43596 (serogroup C) were originally obtained from the ATCC. These two strains were selected because they represent two of the most commonly-occurring serogroups isolated from patients with antibiotic-associated pseudomembranous colitis. [Delmee et al., J. Clin. Microbiol., 28(10):2210 (1990).] Additionally, both of these strains have been previously characterized with respect to their virulence in the Syrian hamster model forC. difficileinfection. [Delmee et al., J. Med Microbiol., 33:85 (1990).]

The bacterial strains were separately cultured on brain heart infusion agar for 48 hours at 37° C. in a[0184]Gas Pack 100 Jar (BBL, Cockeysville, Md.) equipped with a Gas Pack Plus anaerobic envelope (BBL). Forty-eight hour cultures were used because they produce better growth and the organisms have been found to be more cross-reactive with respect to their surface antigen presentation. The greater the degree of cross-reactivity of our IgY preparations, the better the probability of a broad range of activity against different strains/serogroups. [Toma et al., J. Clin. Microbiol., 26(3):426 (1988).]

The resulting organisms were removed from the agar surface using a sterile dacron-tip swab, and were suspended in a solution containing 0.4% formaldehyde in PBS, pH 7.2. This concentration of formaldehyde has been reported as producing good results for the purpose of preparing whole-organism immunogen suspensions for the generation of polyclonal anti-[0185]C. difficileantisera in rabbits. [Delmee et al., J. Clin. Microbiol., 21:323 (1985); Davies et al., Microbial Path., 9:141 (1990).] In this manner, two separate bacterial suspensions were prepared, one for each strain. The two suspensions were then incubated at 4° C. for 1 hour. Following this period of formalin-treatment, the suspensions were centrifuged at 4,200×g for 20 min., and the resulting pellets were washed twice in normal saline. The washed pellets, which contained formalin-treated whole organisms, were resuspended in fresh normal saline such that the visual turbidity of each suspension corresponded to a #7 McFarland standard. [M. A. C. Edelstein, “Processing Clinical Specimens for Anaerobic Bacteria: Isolation and Identification Procedures,” in S. M. Finegold et al (eds.)., Bailey andScott's Diagnostic Microbiology, pp. 477-507, C. V. Mosby Co., (1990). The preparation of McFarland nephelometer standards and the corresponding approximate number of organisms for each tube are described in detail at pp. 172-173 of this volume.] Each of the two #7 suspensions was then split into two separate volumes. One volume of each suspension was volumetrically adjusted, by the addition of saline, to correspond to the visual turbidity of a #1 McFarland standard. [Id.] The #1 suspensions contained approximately 3×10⁸organisms/ml, and the #7 suspensions contained approximately 2×10⁹organisms/ml. [Id.] The four resulting concentration-adjusted suspensions of formalin-treatedC. difficileorganisms were considered to be “bacterial immunogen suspensions.” These suspensions were used immediately after preparation for the initial immunization. [See section (b).]

The formalin-treatment procedure did not result in 100% non-viable bacteria in the immunogen suspensions. In order to increase the level of killing, the formalin concentration and length of treatment were both increased for subsequent immunogen preparations, as described below in Table 3. (Although viability was decreased with the stronger formalin treatment, 100% inviability of the bacterial immunogen suspensions was not reached.) Also, in subsequent immunogen preparations, the formalin solutions were prepared in normal saline instead of PBS. At day 49, the day of the fifth immunization, the excess volumes of the four previous bacterial immunogen suspensions were stored frozen at −70° C. for use during all subsequent immunizations.[0186]

b) Immunization[0187]

For the initial immunization, 1.0 ml volumes of each of the four bacterial immunogen suspensions described above were separately emulsified in 1.2 ml volumes of CFA (GIBCO). For each of the four emulsified immunogen suspensions, two four-month old White Leghorn hens (pre-laying) were immunized. (It is not necessary to use pre-laying hens; actively-laying hens can also be utilized.) Each hen received a total volume of approximately 1.0 ml of a single emulsified immunogen suspension via four injections (two subcutaneous and two intramuscular) of approximately 250 μl per site. In this manner, a total of four different immunization combinations, using two hens per combination, were initiated for the purpose of evaluating both the effect of immunizing concentration on egg yolk antibody (IgY) production, and interstrain cross-reactivity of IgY raised against heterologous strains. The four immunization groups are summarized in Table 3.[0188]

TABLE 3


Immunization Groups

Group
Designation	Immunizing Strain	Approximate Immunizing Dose

CD 43594, #1	C. difficile	1.5 × 10⁸organisms/hen
	strain 43594
CD 43594, #7	C. difficile	1.0 × 10⁹organisms/hen
	strain 43594
CD 43596, #1	C. difficile	1.5 × 10⁸organisms/hen
	strain 43596
CD 43596, #7	C. difficile	1.0 × 10⁹organisms/hen
	strain 43596

The time point for the first series of immunizations was designated as “day zero.” All subsequent immunizations were performed as described above except that the bacterial immunogen suspensions were emulsified using IFA (GIBCO) instead of CFA, and for the later time point immunization, the stored frozen suspensions were used instead of freshly-prepared suspensions. The immunization schedule used is listed in Table 4.[0189]

TABLE 4


Immunization Schedule

Day Of
Immunization	Formalin-Treatment	Immunogen Preparation Used

0	1%, 1 hr.	freshly-prepared
14	1%, overnight	″
21	1%, overnight	″
35	1%, 48 hrs.	″
49	1%, 72 hrs.	″
70	″	stored frozen
85	″	″
105	″	″

c) Purification of Anti-Bacterial Chicken Antibodies[0190]

Groups of four eggs were collected per immunization group between[0191]days 80 and 84 post-initial immunization, and chicken immunoglobulin (IgY) was extracted according to a modification of the procedure of A. Polson et al., Immunol. Comm., 9:495 (1980). A gentle stream of distilled water from a squirt bottle was used to separate the yolks from the whites, and the yolks were broken by dropping them through a funnel into a graduated cylinder. The four individual yolks were pooled for each group. The pooled, broken yolks were blended with 4 volumes of egg extraction buffer to improve antibody yield (egg extraction buffer is 0.01 M sodium phosphate, 0.1 M NaCl, pH 7.5, containing 0.005% thimerosal), and PEG 8000 (Amresco) was added to a concentration of 3.5%. When all the PEG dissolved, the protein precipitates that formed were pelleted by centrifugation at 13,000×g for 10 minutes. The supernatants were decanted and filtered through cheesecloth to remove the lipid layer, and the PEG was added to the supernatants to a final concentration of 12% (the supernatants were assumed to contain 3.5% PEG). After a second centrifugation, the supernatants were discarded and the pellets were centrifuged a final time to extrude the remaining PEG. These crude IgY pellets were then dissolved in the original yolk volume of egg extraction buffer and stored at 4° C. As an additional control, a preimmune IgY solution was prepared as described above, using eggs collected from unimmunized hens.

d) Detection of Anti-Bacterial Antibodies in the Purified IgY Preparations[0192]

In order to evaluate the relative levels of specific anti-[0193]C. difficileactivity in the IgY preparations described above, a modified version of the whole-organism ELISA procedure of N. V. Padhye et al., J. Clin. Microbiol. 29:99-103 (1990) was used. Frozen organisms of bothC difficilestrains described above were thawed and diluted to a concentration of approximately 1×10⁷organisms/ml using PBS, pH 7.2. In this way, two separate coating suspensions were prepared, one for each immunizing strain. Into the wells of 96-well microtiter plates (Falcon, Pro-Bind Assay Plates) were placed 100 μl volumes of the coating suspensions. In this manner, each plate well received a total of approximately 1×10⁶organisms of one strain or the other. The plates were then incubated at 4° C. overnight. The next morning, the coating suspensions were decanted, and all wells were washed three times using PBS. In order to block non-specific binding sites, 100 μl of 0.5% BSA (Sigma) in PBS was then added to each well, and the plates were incubated for 2 hours at room temperature. The blocking solution was decanted, and 100 μl volumes of the IgY preparations described above were initially diluted 1:500 with a solution of 0.1% BSA in PBS, and then serially diluted in 1:5 steps. The following dilutions were placed in the wells: 1:500, 1:2,500, 1:62,5000, 1:312,500, and 1:1,562,500. The plates were again incubated for 2 hours at room temperature. Following this incubation, the IgY-containing solutions were decanted, and the wells were washed three times using BBS-Tween (0.1 M boric acid, 0.025 M sodium borate, 1.0 M NaCl, 0.1% Tween-20), followed by two washes using PBS-Tween (0.1% Tween-20), and finally, two washes using PBS only. To each well, 100 μl of a 1:750 dilution of rabbit anti-chicken IgG (whole-molecule)-alkaline phosphatase conjugate (Sigma) (diluted in 0.1% BSA in PBS) was added. The plates were again incubated for 2 hours at room temperature. The conjugate solutions were decanted and the plates were washed as described above, substituting 50 mM Na₂CO₃, pH 9.5 for the PBS in the final wash. The plates were developed by the addition of 100 μl of a solution containing 1 mg/ml para-nitrophenyl phosphate (Sigma) dissolved in 50 mM Na₂CO₃, 10 mM MgCl₂, pH 9.5 to each well, and incubating the plates at room temperature in the dark for 45 minutes. The absorbance of each well was measured at 410 nm using a Dynatech MR 700 plate reader. In this manner, each of the four IgY preparations described above was tested for reactivity against both of the immunizingC. difficilestrains; strain-specific, as well as cross-reactive activity was determined.

Table 5 shows the results of the whole-organism ELISA. All four IgY preparations demonstrated significant levels of activity, to a dilution of 1:62,500 or greater against both of the immunizing organism strains. Therefore, antibodies raised against one strain were highly cross-reactive with the other strain, and vice versa. The immunizing concentration of organisms did not have a significant effect on organism-specific IgY production, as both concentrations produced approximately equivalent responses. Therefore, the lower immunizing concentration of approximately 1.5×10[0194]⁸organisms/hen is the preferred immunizing concentration of the two tested. The preimmune IgY preparation appeared to possess relatively low levels ofC. difficile-reactive activity to a dilution of 1:500, probably due to prior exposure of the animals to environmental clostridia.

An initial whole-organism ELISA was performed using IgY preparations made from single CD 43594, #1 and CD 43596, #1 eggs collected around day 50 (data not shown). Specific titers were found to be 5 to 10-fold lower than those reported in Table 5. These results demonstrate that it is possible to begin immunizing hens prior to the time that they begin to lay eggs, and to obtain high titer specific IgY from the first eggs that are laid. In other words, it is not necessary to wait for the hens to begin laying before the immunization schedule is started.[0195]

TABLE 5


Results Of The Anti-C. difficileWhole-Organism ELISA

	Dilution Of	43594-Coated
IgY Preparation	IgY Prep	Wells	43596-Coated Wells

CD 43594, #1	1:500	1.746	1.801
	1:2,500	1.092	1.670
	1:12,500	0.202	0.812
	1:62,500	0.136	0.179
	1:312,500	0.012	0.080
	1:1,562,500	0.002	0.020
CD 43594, #7	1:500	1.780	1.771
	1:2,500	1.025	1.078
	1:12,500	0.188	0.382
	1:62,500	0.052	0.132
	1:312,500	0.022	0.043
	1:1,562,500	0.005	0.024
CD 43596, #1	1:500	1.526	1.790
	1:2,500	0.832	1.477
	1:12,500	0.247	0.452
	1:62,500	0.050	0.242
	1:312,500	0.010	0.067
	1:1,562,500	0.000	0.036
CD 43596, #7	1:500	1.702	1.505
	1:2,500	0.706	0.866
	1:12,500	0.250	0.282
	1:62,500	0.039	0.078
	1:312,500	0.002	0.017
	1:1,562,500	0.000	0.010
Preimmune IgY	1:500	0.142	0.309
	1:2,500	0.032	0.077
	1:12,500	0.006	0.024
	1:62,500	0.002	0.012
	1:312,500	0.004	0.010
	1:1,562,500	0.002	0.014

Example 2Treatment ofC. difficileInfection with Anti-C. difficileAntibody

In order to determine whether the immune IgY antibodies raised against whole[0196]C. difficileorganisms were capable of inhibiting the infection of hamsters byC. difficile, hamsters infected by these bacteria were utilized. [Lyerly et al., Infect. Immun., 59:2215-2218 (1991).] This example involved: (a) determination of the lethal dose ofC. difficileorganisms; and (b) treatment of infected animals with immune antibody or control antibody in nutritional solution.

a) Determination of the Lethal Dose of[0197]C. difricileOrganisms

Determination of the lethal dose of[0198]C. difficileorganisms was carried out according to the model described by D. M. Lyerly et al., Infect. Immun., 59:2215-2218 (1991).C. difficilestrain ATCC 43596 (serogroup C, ATCC) was plated on BHI agar and grown anaerobically (BBL Gas Pak 100 system) at 37° C. for 42 hours. Organisms were removed from the agar surface using a sterile dacron-tip swab and suspended in sterile 0.9% NaCl solution to a density of 10⁸organisms/ml.

In order to determine the lethal dose of[0199]C. difficilein the presence of control antibody and nutritional formula, non-immune eggs were obtained from unimmunized hens and a 12% PEG preparation made as described in Example 1(c). This preparation was redissolved in one fourth the original yolk volume of vanilla flavor Ensure®.

Starting on day one, groups of female Golden Syrian hamsters (Harlan Sprague Dawley), 8-9 weeks old and weighing approximately 100 gm, were orally administered 1 ml of the preimmune/Ensure® formula at time zero, 2 hours, 6 hours, and 10 hours. At 1 hour, animals were orally administered 3.0 mg clindamycin HCl (Sigma) in 1 ml of water. This drug predisposes hamsters to[0200]C. difficileinfection by altering the normal intestinal flora. On day two, the animals were given 1 ml of the preimmune IgY/Ensure® formula at time zero, 2 hours, 6 hours, and 10 hours. At 1 hour on day two, different groups of animals were inoculated orally with saline (control), or 10², 10⁴, 10⁶, or 10° C. dificile organisms in 1 ml of saline. From days 3-12, animals were given 1 ml of the preimmune IgY/Ensure® formula three times daily and observed for the onset of diarrhea and death. Each animal was housed in an individual cage and was offered food and water ad libitum.

Administration of 10[0201]⁶-10⁸organisms resulted in death in 3-4 days while the lower doses of 10²-10⁴organisms caused death in 5 days. Cecal swabs taken from dead animals indicated the presence ofC. difficile. Given the effectiveness of the 10²dose, this number of organisms was chosen for the following experiment to see if hyperimmune anti-C. difficileantibody could block infection.

b) Treatment of Infected Animals with Immune Antibody or Control Antibody in Nutritional Formula[0202]

The experiment in (a) was repeated using three groups of seven hamsters each. Group A received no clindamycin or[0203]C. difficileand was the survival control. Group B received clindamycin, 10²C. difficileorganisms and preimmune IgY on the same schedule as the animals in (a) above. Group C received clindamycin, 102C. difficileorganisms, and hyperimmune anti-C. difficileIgY on the same schedule as Group B. The anti-C. difficileIgY was prepared as described in Example 1 except that the 12% PEG preparation was dissolved in one fourth the original yolk volume of Ensure®.

All animals were observed for the onset of diarrhea or other disease symptoms and death. Each animal was housed in an individual cage and was offered food and water ad libitum. The results are shown in Table 6.[0204]

TABLE 6


The Effect Of Oral Feeding Of Hyperimmune
IgY Antibody onC. difficileInfection

	Time To	Time To
Animal Group	Diarrhea¹	Death¹

A pre-immune IgY only	no diarrhea	no deaths
B Clindamycin,C. difficile,preimmune IgY	30 hrs.	49 hrs.
C Clindamycin,C. difficile, immune IgY	33 hrs.	56 hrs.

Hamsters in the control group A did not develop diarrhea and remained healthy during the experimental period. Hamsters in groups B and C developed diarrheal disease. Anti-[0205]C. difficile[gY did not protect the animals from diarrhea or death, all animals succumbed in the same time interval as the animals treated with preimmune IgY. Thus, while immunization with whole organisms apparently can improve sub-lethal symptoms with particular bacteria (see U.S. Pat. No. 5,080,895 to H. Tokoro), such an approach does not prove to be productive to protect against the lethal effects ofC. difficile.

Example 3Production ofC. botulinumType A Antitoxin in Hens

In order to determine whether antibodies could be raised against the toxin produced by clostridial pathogens, which would be effective in treating clostridial diseases, antitoxin to[0206]C. botulinumtype A toxin was produced. This example involves: (a) toxin modification; (b) immunization; (c) antitoxin collection; (d) antigenicity assessment; and (e) assay of antitoxin titer.

a) Toxin Modification[0207]

[0208]C. botulinumtype A toxoid was obtained from B. R. DasGupta. From this, the active type A neurotoxin (M.W. approximately 150 kD) was purified to greater than 99% purity, according to published methods. (B. R. DasGupta & V. Sathyamoorthy, Toxicon, 22:415 (1984).] The neurotoxin was detoxified with formaldehyde according to published methods. [B. R. Singh & B. R. DasGupta, Toxicon, 27:403 (1989).]

b) Immunization[0209]

[0210]C. botulinumtoxoid for immunization was dissolved in PBS (1 mg/ml) and was emulsified with an approximately equal volume of CFA (GIBCO) for initial immunization or IFA for booster immunization. On day zero, two white leghorn hens, obtained from local breeders, were each injected at multiple sites (intramuscular and subcutaneous) with 1 ml inactivated toxoid emulsified in 1 ml CFA. Subsequent booster immunizations were made according to the following schedule for day of injection and toxoid amount:days 14 and 21-0.5 mg; day 171-0.75 mg; days 394, 401, 409-0.25 mg. One hen received an additional booster of 0.150 mg on day 544.

c) Antitoxin Collection[0211]

Total yolk immunoglobulin (IgY) was extracted as described in Example 1(c) and the IgY pellet was dissolved in the original yolk volume of PBS with thimerosal.[0212]

d) Antigenicity Assessment[0213]

Eggs were collected from day 409 through day 423 to assess whether the toxoid was sufficiently immunogenic to raise antibody. Eggs from the two hens were pooled and antibody was collected as described in the standard PEG protocol. [Exanple 1(c).] Antigenicity of the botulinal toxin was assessed on Western blots. The 150 kD detoxified type A neurotoxin and unmodified, toxic, 300 kD botulinal type A complex (toxin used for intragastric route administration for animal gut neutralization experiments; see Example 6) were separated on a SDS-polyacrylamide reducing gel. The Western blot technique was performed according to the method of Towbin. [H. Towbin et al., Proc. Natl. Acad. Sci. USA, 76:4350 (1979).] Ten μg samples of[0214]C. botulinumcomplex and toxoid were dissolved in SDS reducing sample buffer (1% SDS, 0.5% 2-mercaptoethanol, 50 mM Tris, pH 6.8, 10% glycerol, 0.025% w/v bromphenol blue, 10% β-mercaptoethanol), heated at 95° C. for 10 min and separated on a 1 mm thick 5% SDS-polyacrylamide gel. [K. Weber and M. Osborn, “Proteins and Sodium Dodecyl Sulfate: Molecular Weight Determination on Polyacrylamide Gels and Related Procedures,” in The Proteins, 3d Edition (H. Neurath & R. L. Hill, eds), pp. 179-223, (Academic Press, NY, 1975).] Part of the gel was cut off and the proteins were stained with Coomassie Blue. The proteins in the remainder of the gel were transferred to nitrocellulose using the Milliblot-SDE electro-blotting system (Millipore) according to manufacturer's directions. The nitrocellulose was temporarily stained with 10% Ponceau S [S. B. Carroll and A. Laughon, “Production and Purification of Polyclonal Antibodies to the Foreign Segment ofβ-galactosidase Fusion Proteins,” in DNA Cloning: A Practical Approach, Vol.III, (D. Glover, ed.), pp. 89-111, IRL Press, Oxford, (1987)] to visualize the lanes, then destained by running a gentle stream of distilled water over the blot for several minutes. The nitrocellulose was immersed in PBS containing 3% BSA overnight at 4° C. to block any remaining protein binding sites.

The blot was cut into strips and each strip was incubated with the appropriate primary antibody. The avian anti-[0215]C. botulinumantibodies [described in (c)] and pre-immune chicken antibody (as control) were diluted 1:125 in PBS containing 1 mg/ml BSA for 2 hours at room temperature. The blots were washed with two changes each of large volumes of PBS, BBS-Tween and PBS, successively (10 min/wash). Goat anti-chicken IgG alkaline phosphatase conjugated secondary antibody (Fisher Biotech) was diluted 1:500 in PBS containing 1 mg/ml BSA and incubated with the blot for 2 hours at room temperature. The blots were washed with two changes each of large volumes of PBS and BBS-Tween, followed by one change of PBS and 0.1 M Tris-HCl, pH 9.5. Blots were developed in freshly prepared alkaline phosphatase substrate buffer (100 μg/ml nitroblue tetrazolium (Sigma), 50 μg/ml 5-bromo-4-chloro-3-indolyl phosphate (Sigma), 5 mM MgCl₂in 50 mM Na₂CO₃, pH 9.5).

The Western blots are shown in FIG. 1. The anti-[0216]C. botulinumIgY reacted to the toxoid to give a broad immunoreactive band at about 145-150 kD on the reducing gel. This toxoid is refractive to disulfide cleavage by reducing agents due to formalin crosslinking. The immune IgY reacted with the active toxin complex, a 97 kDC. botulinumtype A heavy chain and a 53 kD light chain. The preimmune IgY was unreactive to theC. botulinumcomplex or toxoid in the Western blot.

e) Antitoxin Antibody Titer[0217]

The IgY antibody titer to[0218]C. botulinumtype A toxoid of eggs harvested between day 409 and 423 was evaluated by ELISA, prepared as follows. Ninety-six-well Falcon Pro-bind plates were coated overnight at 4° C. with 100 μl/well toxoid [B. R. Singh & B. R. Das Gupta, Toxicon 27:403 (1989)] at 2.5 μg/ml in PBS, pH 7.5 containing 0.005% thimerosal. The following day the wells were blocked with PBS containing 1% BSA for 1 hour at 37° C. The IgY from immune or preimmune eggs was diluted in PBS containing 1% BSA and 0.05% Tween 20 and the plates were incubated for 1 hour at 37° C. The plates were washed three times with PBS containing 0.05% Tween 20 and three times with PBS alone. Alkaline phosphatase-conjugated goat-anti-chicken IgG (Fisher Biotech) was diluted 1:750 in PBS containing 1% BSA and 0.05% Tween 20, added to the plates, and incubated 1 hour at 37° C. The plates were washed as before, and p-nitrophenyl phosphate (Sigma) at 1 mg/ml in 0.05 M Na₂CO₃, pH 9.5, 10 mM MgCl₂was added.

The results are shown in FIG. 2. Chickens immunized with the toxoid generated high titers of antibody to the immunogen. Importantly, eggs from both immunized hens had significant anti-immunogen antibody titers as compared to preimmune control eggs. The anti-[0219]C. botulinumIgY possessed significant activity, to a dilution of 1:93,750 or greater.

Example 4Preparation of Avian Egg Yolk Immunoglobulin in an Orally Administrable Form

In order to administer avian IgY antibodies orally to experimental mice, an effective delivery formula for the IgY had to be determined. The concern was that if the crude IgY was dissolved in PBS, the saline in PBS would dehydrate the mice, which might prove harmful over the duration of the study. Therefore, alternative methods of oral administration of IgY were tested. The example involved: (a) isolation of immune IgY; (b) solubilization of IgY in water or PBS, including subsequent dialysis of the IgY-PBS solution with water to eliminate or reduce the salts (salt and phosphate) in the buffer; and (c) comparison of the quantity and activity of recovered IgY by absorbance at 280 nm and PAGE, and enzyme-linked immunoassay (ELISA).[0220]

a) Isolation of Immune IgY[0221]

In order to investigate the most effective delivery formula for IgY, we used IgY which was raised against[0222]Crotalus durissus terrificusvenom. Three eggs were collected from hens immunized with theC. durissus terrificusvenom and IgY was extracted from the yolks using the modified Polson procedure described by Thalley and Carroll [Bio/Technology, 8:934-938 (1990)] as described in Example 1(c).

The egg yolks were separated from the whites, pooled, and blended with four volumes of PBS. Powdered PEG 8000 was added to a concentration of 3.5%. The mixture was centrifuged at 10,000 rpm for 10 minutes to pellet the precipitated protein, and the supernatant was filtered through cheesecloth to remove the lipid layer. Powdered PEG 8000 was added to the supernatant to bring the final PEG concentration to 12% (assuming a PEG concentration of 3.5% in the supernatant). The 12% PEG/IgY mixture was divided into two equal volumes and centrifuged to pellet the IgY.[0223]

b) Solubilization of the IgY in Water or PBS[0224]

One pellet was resuspended in ½ the original yolk volume of PBS, and the other pellet was resuspended in ½ the original yolk volume of water. The pellets were then centrifuged to remove any particles or insoluble material. The IgY in PBS solution dissolved readily but the fraction resuspended in water remained cloudy.[0225]

In order to satisfy anticipated sterility requirements for orally administered antibodies, the antibody solution needs to be filter-sterilized (as an alternative to heat sterilization which would destroy the antibodies). The preparation of IgY resuspended in water was too cloudy to pass through either a 0.2 or 0.45 μm membrane filter, so 10 ml of the PBS resuspended fraction was dialyzed overnight at room temperature against 250 ml of water. The following morning the dialysis chamber was emptied and refilled with 250 ml of fresh H[0226]₂O for a second dialysis. Thereafter, the yields of soluble antibody were determined at OD₂₈₀and are compared in Table 7.

TABLE 7


Dependence Of IgY Yield On Solvents

	Absorbance Of 1:10
Fraction	Dilution At 280 nm	Percent Recovery

PBS dissolved	1.149	100%
H₂O dissolved	0.706	61%
PBS dissolved/H₂O dialyzed	0.885	77%

Resuspending the pellets in PBS followed by dialysis against water recovered more antibody than directly resuspending the pellets in water (77% versus 61%). Equivalent volumes of the IgY preparation in PBS or water were compared by PAGE, and these results were in accordance with the absorbance values (data not shown).[0227]

c) Activity of IgY Prepared with Different Solvents[0228]

An ELISA was performed to compare the binding activity of the IgY extracted by each procedure described above.[0229]C. durissus terrificus(C.d t.) venom at 2.5 μg/ml in PBS was used to coat each well of a 96-well microtiter plate. The remaining protein binding sites were blocked with PBS containing 5 mg/ml BSA. Primary antibody dilutions (in PBS containing 1 mg/ml BSA) were added in duplicate. After 2 hours of incubation at room temperature, the unbound primary antibodies were removed by washing the wells with PBS, BBS-Tween, and PBS. The species specific secondary antibody (goat anti-chicken immunoglobulin alkaline-phosphatase conjugate (Sigma) was diluted 1:750 in PBS containing 1 mg/ml BSA and added to each well of the microtiter plate. After 2 hours of incubation at room temperature, the unbound secondary antibody was removed by washing the plate as before, and freshly prepared alkaline phosphatase substrate (Sigma) at 1 mg/ml in 50 mM Na₂CO₃, 10 mM MgCl₂, pH 9.5 was added to each well. The color development was measured on a Dynatech MR 700 microplate reader using a 412 nm filter. The results are shown in Table 8.

The binding assay results parallel the recovery values in Table 7, with PBS-dissolved IgY showing slightly more activity than the PBS-dissolved/H[0230]₂O dialyzed antibody. The water-dissolved antibody had considerably less binding activity than the other preparations.

Example 5Survival of Antibody Activity After Passage Through the Gastrointestinal Tract

In order to determine the feasibility of oral administration of antibody, it was of interest to determine whether orally administered IgY survived passage through the gastrointestinal tract. The example involved: (a) oral administration of specific immune antibody mixed with a nutritional formula; and (b) assay of antibody activity extracted from feces.[0231]

TABLE 8


Antigen-Binding Activity Of IgY
Prepared With Different Solvents

Dilution	Preimmune	PBS Dissolved	H₂O Dissolved	PBS/H₂O

1:500	0.005	1.748	1.577	1.742
1:2,500	0.004	0.644	0.349	0.606
1:12,500	0.001	0.144	0.054	0.090
1:62,500	0.001	0.025	0.007	0.016
1:312,500	0.010	0.000	0.000	0.002

a) Oral Administration of Antibody[0232]

The IgY preparations used in this example are the same PBS-dissolved/H[0233]₂O dialyzed antivenom materials obtained in Example 4 above, mixed with an equal volume of Enfamil®. Two mice were used in this experiment, each receiving a different diet as follows:

1) water and food as usual;[0234]

2) immune IgY preparation dialyzed against water and mixed 1:1 with Enfamil®. (The mice were given the corresponding mixture as their only source of food and water).[0235]

b) Antibody Activity After Ingestion[0236]

After both mice had ingested their respective fluids, each tube was refilled with approximately 10 ml of the appropriate fluid first thing in the morning. By mid-morning there was about 4 to 5 ml of liquid left in each tube. At this point stool samples were collected from each mouse, weighed, and dissolved in approximately 500 μl PBS per 100 mg stool sample. One hundred and sixty mg of control stools (no antibody) and 99 mg of experimental stools (specific antibody) in 1.5 ml microfuge tubes were dissolved in 800 and 500 μl PBS, respectively. The samples were heated at 37° C. for 10 minutes and vortexed vigorously. The experimental stools were also broken up with a narrow spatula Each sample was centrifuged for 5 minutes in a microfuge and the supernatants, presumably containing the antibody extracts, were collected. The pellets were saved at 2-8° C. in case future extracts were needed. Because the supernatants were tinted, they were diluted five-fold in PBS containing 1 mg/ml BSA for the initial dilution in the enzyme immunoassay (ELISA). The primary extracts were then diluted five-fold serially from this initial dilution. The volume of primary extract added to each well was 190 μl. The ELISA was performed exactly as described in Example 4.[0237]

TABLE 9


Specific Antibody Activity After Passage
Through The Gastrointestinal Tract

Dilution	Preimmune IgY	Control Fecal Extract	EXP. Fecal Extract

1:5	<0	0.000	0.032
1:25	0.016	<0	0.016
1:125	<0	<0	0.009
1:625	<0	0.003	0.001
1:3125	<0	<0	0.000

There was some active antibody in the fecal extract from the mouse given the specific antibody in Enfamil® formula, but it was present at a very low level. Since the samples were assayed at an initial 1:5 dilution, the binding observed could have been higher with less dilute samples. Consequently, the mice were allowed to continue ingesting either regular food and water or the specific IgY in Enfamil® formula, as appropriate, so the assay could be repeated. Another ELISA plate was coated overnight with 5 μg/ml of C.d.t. venom in PBS.[0238]

The following morning the ELISA plate was blocked with 5 mg/ml BSA, and the fecal samples were extracted as before, except that instead of heating the extracts at 37° C., the samples were kept on ice to limit proteolysis. The samples were assayed undiluted initially, and in 5× serial dilutions thereafter. Otherwise the assay was carried out as before.[0239]

TABLE 10


Specific Antibody Survives Passage
Through The Gastrointestinal Tract

	Dilution	Preimmune IgY	Control Extract	Exp. Extract

undiluted	0.003	<0	0.379
1:5	<0	<0	0.071
1:25	0.000	<0	0.027
1:125	0.003	<0	0.017
1:625	0.000	<0	0.008
1:3125	0.002	<0	0.002

The experiment confirmed the previous results, with the antibody activity markedly higher. The control fecal extract showed no anti-C.d.t. activity, even undiluted, while the fecal extract from the anti-C.d.t. IgY/Enfamil®-fed mouse showed considerable anti-C.d.t. activity. This experiment (and the previous experiment) clearly demonstrate that active IgY antibody survives passage through the mouse digestive tract, a finding with favorable implications for the success of IgY antibodies administered orally as a therapeutic or prophylactic.[0240]

Example 6In Vivo Neutralization of TypeC. botulinumType A Neurotoxin by Avian Antitoxin Antibody

This example demonstrated the ability of PEG-purified antitoxin, collected as described in Example 3, to neutralize the lethal effect of[0241]C. botulinumneurotoxin type A in mice. To determine the oral lethal dose (LD₁₀₀) of toxin A, groups of BALB/c mice were given different doses of toxin per unit body weight (average body weight of 24 grams). For oral administration, toxin A complex, which contains the neurotoxin associated with other non-toxin proteins was used. This complex is markedly more toxic than purified neurotoxin when given by the oral route. [I. Ohishi et al., Infect. Immun., 16:106 (1977).]C. botulinumtoxin type A complex, obtained from Eric Johnson (University Of Wisconsin, Madison) was 250 μg/ml in 50 mM sodium citrate, pH 5.5,specific toxicity 3×10⁷mouse LD₅O/mg with parenteral administration. Approximately 40-50 ng/gm body weight was usually fatal within 48 hours in mice maintained on conventional food and water. When mice were given a diet ad libitum of only Enfamil® the concentration needed to produce lethality was approximately 2.5 times higher (125 ng/gm body weight). Botulinal toxin concentrations of approximately 200 ng/gm body weight were fatal in mice fed Enfamil® containing preimmune IgY (resuspended in Enfamil® at the original yolk volume).

The oral LD[0242]₁₀₀ofC. botulinumtoxin was also determined in mice that received known amounts of a mixture of preimmune IgY-Ensure® delivered orally through feeding needles. Using a 22 gauge feeding needle, mice were given 250 μl each of a preimmune IgY-Ensure® mixture (preimmune IgY dissolved in ¼ original yolk volume) 1 hour before and ½ hour and 5 hours after administering botulinal toxin. Toxin concentrations given orally ranged from approximately 12 to 312 ng/gm body weight (0.3 to 7.5 μg per mouse). Botulinal toxin complex concentration of approximately 40 ng/gm body weight (1 μg per mouse) was lethal in all mice in less than 36 hours.

Two groups of BALB/c mice, 10 per group, were each given orally a single dose of 1 μg each of botulinal toxin complex in 100 μl of 50 mM sodium citrate pH 5.5. The mice received 250 μl treatments of a mixture of either preimmune or immune IgY in Ensure® (¼original yolk volume) 1 hour before and ½ hour, 4 hours, and 8 hours after botulinal toxin administration. The mice received three treatments per day for two more days. The mice were observed for 96 hours. The survival and mortality are shown in Table 11.[0243]

TABLE 11


Neutralization Of Botulinal Toxin A In Vivo

Toxin Dose		Number Of	Number Of
ng/gm	Antibody Type	Mice Alive	Mice Dead

41.6	non-immune	0	10
41.6	anti-botulinal toxin	10	0

All mice treated with the preimmune IgY-Ensure®) mixture died within 46 hours post-toxin administration. The average time of death in the mice was 32 hours post toxin administration. Treatments of preimmune IgY-Ensure® mixture did not continue beyond 24 hours due to extensive paralysis of the mouth in mice of this group. In contrast, all ten mice treated with the immune anti-botulinal toxin IgY-Ensure® mixture survived past 96 hours. Only 4 mice in this group exhibited symptoms of botulism toxicity (two mice about 2 days after and two[0244]mice 4 days after toxin administration). These mice eventually died 5 and 6 days later. Six of the mice in this immune group displayed no adverse effects to the toxin and remained alive and healthy long term. Thus, the avian anti-botulinal toxin antibody demonstrated very good protection from the lethal effects of the toxin in the experimental mice.

Example 7Production of an Avian Antitoxin AgainstClostridium difficileToxin A

Toxin A is a potent cytotoxin secreted by pathogenic strains of[0245]C. difficile, that plays a direct role in damaging gastrointestinal tissues. In more severe cases ofC. difficileintoxication, pseudomembranous colitis can develop which may be fatal. This would be prevented by neutralizing the effects of this toxin in the gastrointestinal tract. As a first step, antibodies were produced against a portion of the toxin. The example involved: (a) conjugation of a synthetic peptide of toxin A to bovine serum albumin; (b) immunization of hens with the peptide-BSA conjugate; and (c) detection of antitoxin peptide antibodies by ELISA.

a) Conjugation of a Synthetic Peptide Of Toxin A to Bovine Serum Albumin[0246]

The synthetic peptide CQTIDGKKYYFN-NH[0247]₂(SEQ ID. NO:82) was prepared commercially (Multiple Peptide Systems, San Diego, Calif.) and validated to be >80% pure by high-pressure liquid chromatography. The eleven amino acids following the cysteine residue represent a consensus sequence of a repeated amino acid sequence found in Toxin A. [Wren et al., Infect. Immun., 59:3151-3155 (1991).] The cysteine was added to facilitate conjugation to carrier protein.

In order to prepare the carrier for conjugation, BSA (Sigma) was dissolved in 0.01 M NaPO[0248]₄, pH 7.0 to a final concentration of 20 mg/ml and n-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Pierce) was dissolved in N,N-dimethyl formamide to a concentration of 5 mg/ml. MBS solution, 0.51 ml, was added to 3.25 ml of the BSA solution and incubated for 30 minutes at room temperature with stirring every 5 minutes. The MBS-activated BSA was then purified by chromatography on a Bio-Gel P-10 column (Bio-Rad; 40 ml bed volume) equilibrated with 50 mM NaPO₄, pH 7.0 buffer. Peak fractions were pooled (6.0 ml).

Lyophilized toxin A peptide (20 mg) was added to the activated BSA mixture, stirred until the peptide dissolved and incubated 3 hours at room temperature. Within 20 minutes, the reaction mixture became cloudy and precipitates formed. After 3 hours, the reaction mixture was centrifuged at 10,000×g for 10 min and the supernatant analyzed for protein content. No significant protein could be detected at 280 nm. The conjugate precipitate was washed three times with PBS and stored at 4° C. A second conjugation was performed with 15 mg of activated BSA and 5 mg of peptide and the conjugates pooled and suspended at a peptide concentration of 10 mg/ml in 10 mM NaPO[0249]₄, pH 7.2.

b) Immunization of Hens with Peptide Conjugate[0250]

Two hens were each initially immunized on day zero by injection into two subcutaneous and two intramuscular sites with 1 mg of peptide conjugate that was emulsified in CFA (GIBCO). The hens were boosted on[0251]day 14 and day 21 with 1 mg of peptide conjugate emulsified in IFA (GIBCO).

c) Detection of Antitoxin Peptide Antibodies by ELISA[0252]

IgY was purified from two eggs obtained before immunization (pre-immune) and two eggs obtained 31 and 32 days after the initial immunization using PEG fractionation as described in Example 1.[0253]

Wells of a 96-well microtiter plate (Falcon Pro-Bind Assay Plate) were coated overnight at 4° C. with 100 μg/ml solution of the toxin A synthetic peptide in PBS, pH 7.2 prepared by dissolving 1 mg of the peptide in 1.0 ml of H[0254]₂O and dilution of PBS. The pre-immune and immune IgY preparations were diluted in a five-fold series in a buffer containing 1% PEG 8000 and 0.1% Tween-20 (v/v) in PBS, pH 7.2. The wells were blocked for 2 hours at room temperature with 150 μl of a solution containing 5% (v/v) Carnation® nonfat dry milk and 1% PEG 8000 in PBS, pH 7.2. After incubation for 2 hours at room temperature, the wells were washed, secondary rabbit anti-chicken IgG-alkaline phosphatase (1:750) added, the wells washed again and the color development obtained as described in Example 1. The results are shown in Table 12.

TABLE 12


Reactivity Of IgY With Toxin Peptide

Absorbance At 410 nm

Dilution Of PEG Prep	Preimmune	Immune Anti-Peptide

1:100	0.013	0.253
1:500	0.004	0.039
1:2500	0.004	0.005

Clearly, the immune antibodies contain titers against this repeated epitope of toxin A.[0255]

Example 8Production of Avian Antitoxins AgainstClostridium difficileNative Toxins A and B

To determine whether avian antibodies are effective for the neutralization of[0256]C. difficiletoxins, hens were immunized using nativeC. difficiletoxins A and B. The resulting egg yolk antibodies were then extracted and assessed for their ability to neutralize toxins A and B in vitro. The Example involved (a) preparation of the toxin immunogens, (b) immunization, (c) purification of the antitoxins, and (d) assay of toxin neutralization activity.

a) Preparation of the Toxin Immunogens[0257]

Both[0258]C. difficilenative toxins A and B, andC. difficiletoxoids, prepared by the treatment of the native toxins with formaldehyde, were employed as immunogens.C. difficiletoxoids A and B were prepared by a procedure which was modified from published methods (Ehrich et al., Infect. Immun. 28:1041 (1980). Separate solutions (in PBS) of nativeC. difficiletoxin A and toxin B (Tech Lab) were each adjusted to a concentration of 0.20 mg/ml, and formaldehyde was added to a final concentration of 0.4%. The toxin/formaldehyde solutions were then incubated at 37° C. for 40 hrs. Free formaldehyde was then removed from the resulting toxoid solutions by dialysis against PBS at 4° C. In previously published reports, this dialysis step was not performed. Therefore, free formaldehyde must have been present in their toxoid preparations. The toxoid solutions were concentrated, using a Centriprep concentrator unit (Amicon), to a final toxoid concentration of 4.0 mg/ml. The two resulting preparations were designated as toxoid A and toxoid B.

[0259]C. difficilenative toxins were prepared by concentrating stock solutions of toxin A and toxin B (Tech Lab, Inc), using Centriprep concentrator units (Amicon), to a final concentration of 4.0 mg/ml.

b) Immunization[0260]

The first two immunizations were performed using the toxoid A and toxoid B immunogens described above. A total of 3 different immunization combinations were employed. For the first immunization group, 0.2 ml of toxoid A was emulsified in an equal volume of Titer Max adjuvant (CytRx). Titer Max was used in order to conserve the amount of immunogen used, and to simplify the immunization procedure. This immunization group was designated “CTA.” For the second immunization group, 0.1 ml of toxoid B was emulsified in an equal volume of Titer Max adjuvant. This group was designated “CTB.” For the third immunization group, 0.2 ml of toxoid A was first mixed with 0.2 ml of toxoid B, and the resulting mixture was emulsified in 0.4 ml of Titer Max adjuvant. This group was designated “CTAB.” In this way, three separate immunogen emulsions were prepared, with each emulsion containing a final concentration of 2.0 mg/ml of toxoid A (CTA) or toxoid B (CTB) or a mixture of 2.0 mg/ml toxoid A and 2.0 mg/ml toxoid B (CTAB).[0261]

On[0262]day 0, White Leghorn hens, obtained from a local breeder, were immunized as follows: Group CTA. Four hens were immunized, with each hen receiving 200 μg of toxoid A, via two intramuscular (I.M.) injections of 50 μl of CTA emulsion in the breast area. Group CTB. One hen was immunized with 200 μg of toxoid B, via two I.M. injections of 50 μl of CTB emulsion in the breast area. Group CTAB. Four hens were immunized, with each hen receiving a mixture containing 200 μg of toxoid A and 200 μg of toxoid B, via two I.M. injections of 100 μl of CTAB emulsion in the breast area. The second immunization was performed 5 weeks later, on day 35, exactly as described for the first immunization above.

In order to determine whether hens previously immunized with[0263]C. difficiletoxoids could tolerate subsequent booster immunizations using native toxins, a single hen from group CTAB was immunized for a third time, this time using a mixture of the native toxin A and native toxin B described in section (a) above (these toxins were not formaldehyde-treated, and were used in their active form). This was done in order to increase the amount (titer) and affinity of specific antitoxin antibody produced by the hen over that achieved by immunizing with toxoids only. On day 62, 0.1 ml of a toxin mixture was prepared which contained 200 μg of native toxin A and 200 μg of native toxin B. This toxin mixture was then emulsified in 0.1 ml of Titer Max adjuvant. A single CTAB hen was then immunized with the resulting immunogen emulsion, via two I.M. injections of 100 μl each, into the breast area. This hen was marked with a wing band, and observed for adverse effects for a period of approximately 1 week, after which time the hen appeared to be in good health.

Because the CTAB hen described above tolerated the booster immunization with native toxins A and B with no adverse effects, it was decided to boost the remaining hens with native toxin as well. On[0264]day 70, booster immunizations were performed as follows: Group CTA. A 0.2 ml volume of the 4 mg/ml native toxin A solution was emulsified in an equal volume of Titer Max adjuvant. Each of the 4 hens was then immunized with 200 μg of native toxin A, as described for the toxoid A immunizations above. Group CTB. A 50 μl volume of the 4 mg/ml native toxin B solution was emulsified in an equal volume of Titer Max adjuvant. The hen was then immunized with 200 μg of native toxin B, as described for the toxoid B immunizations above. Group CTAB. A 0.15 ml volume of the 4 mg/ml native toxin A solution was first mixed with a 0.15 ml volume the 4 mg/ml native toxin B solution. The resulting toxin mixture was then emulsified in 0.3 ml of Titer Max adjuvant. The 3 remaining hens (the hen with the wing band was not immunized this time) were then immunized with 200 μg of native toxin A and 200 μg of native toxin B as described for the toxoid A+ toxoid B immunizations (CTAB) above. On day 85, all hens received a second booster immunization using native toxins, done exactly as described for the first boost with native toxins above.

All hens tolerated both booster immunizations with native toxins with no adverse effects. As previous literature references describe the use of formaldehyde-treated toxoids, this is apparently the first time that any immunizations have been performed using native[0265]C. difficiletoxins.

c) Purification of Antitoxins[0266]

Eggs were collected from the hen in group CTB 10-12 days following the second immunization with toxoid (day 35 immunization described in section (b) above), and from the hens in groups CTA and CTAB 20-21 days following the second immunization with toxoid. To be used as a pre-immune (negative) control, eggs were also collected from unimmunized hens from the same flock. Egg yolk immunoglobulin (IgY) was extracted from the 4 groups of eggs as described in Example 1(c), and the final IgY pellets were solubilized in the original yolk volume of PBS without thimerosal. Importantly, thimerosal was excluded because it would have been toxic to the CHO cells used in the toxin neutralization assays described in section (d) below.[0267]

d) Assay of Toxin Neutralization Activity[0268]

The toxin neutralization activity of the IgY solutions prepared in section (c) above was determined using an assay system that was modified from published methods. [Ehrich et al., Infect. Immun. 28:1041-1043 (1992); and McGee et al. Microb. Path. 12:333-341 (1992).] As additional controls, affinity-purified goat anti-[0269]C. difficiletoxin A (Tech Lab) and affinity-purified goat anti-C. difficiletoxin B (Tech Lab) were also assayed for toxin neutralization activity.

The IgY solutions and goat antibodies were serially diluted using[0270]F 12 medium (GIBCO) which was supplemented with 2% FCS (G[BCO)(this solution will be referred to as “medium” for the remainder of this Example). The resulting antibody solutions were then mixed with a standardized concentration of either nativeC. difficiletoxin A (Tech Lab), or nativeC. difficiletoxin B (Tech Lab), at the concentrations indicated below. Following incubation at 37° C. for 60 min., 100 μl volumes of the toxin+antibody mixtures were added to the wells of 96-well microtiter plates (Falcon Microtest III) which contained 2.5×10⁴Chinese Hamster Ovary (CHO) cells per well (the CHO cells were plated on the previous day to allow them to adhere to the plate wells). The final concentration of toxin, or dilution of antibody indicated below refers to the final test concentration of each reagent present in the respective microtiter plate wells. Toxin reference wells were prepared which contained CHO cells and toxin A or toxin B at the same concentration used for the toxin plus antibody mixtures (these wells contained no antibody). Separate control wells were also prepared which contained CHO cells and medium only. The assay plates were then incubated for 18-24 hrs. in a 37° C., humidified, 5% CO₂incubator. On the following day, the remaining adherent (viable) cells in the plate wells were stained using 0.2% crystal violet (Mallinckrodt) dissolved in 2% ethanol, for 10 min. Excess stain was then removed by rinsing with water, and the stained cells were solubilized by adding 100 μl of 1% SDS (dissolved in water) to each well. The absorbance of each well was then measured at 570 nm, and the percent cytotoxicity of each test sample or mixture was calculated using the following formula: $% CHO Cell Cytotoxicity = [1 - (\frac{Abs . Sample}{Abs . Sample})] \times 100$
Unlike previous reports which quantitate results visually by counting cell rounding by microscopy, this Example utilized spectrophotometric methods to quantitate the[0271]C. difficiletoxin bioassay. In order to determine the toxin A neutralizing activity of the CTA, CTAB, and pre-immune IgY preparations, as well as the affinity-purified goat antitoxin A control, dilutions of these antibodies were reacted against a 0.1 μg/ml concentration of native toxin A (this is the approx. 50% cytotoxic dose of toxin A in this assay system). The results are shown in FIG. 3.
Complete neutralization of toxin A occurred with the CTA IgY (antitoxin A, above) at dilutions of 1:80 and lower, while significant neutralization occurred out to the 1:320 dilution. The CTAB IgY (antitoxin A+toxin B, above) demonstrated complete neutralization at the 1:320-1:160 and lower dilutions, and significant neutralization occurred out to the 1:1280 dilution. The commercially available affinity-purified goat antitoxin A did not completely neutralize toxin A at any of the dilutions tested, but demonstrated significant neutralization out to a dilution of 1:1,280. The preimmune IgY did not show any toxin A neutralizing activity at any of the concentrations tested. These results demonstrate that IgY purified from eggs laid by hens immunized with toxin A alone, or simultaneously with toxin A and toxin B, is an effective toxin A antitoxin.[0272]
The toxin B neutralizing activity of the CTAB and pre-immune IgY preparations, and also the affinity-purified goat antitoxin B control was determined by reacting dilutions of these antibodies against a concentration of native toxin B of 0.1 ng/ml (approximately the 50% cytotoxic dose of toxin B in the assay system). The results are shown in FIG. 4.[0273]
Complete neutralization of toxin B occurred with the CTAB IgY (antitoxin A+toxin B, above) at the 1:40 and lower dilutions, and significant neutralization occurred out to the 1:320 dilution. The affinity-purified goat antitoxin B demonstrated complete neutralization at dilutions of 1:640 and lower, and significant neutralization occurred out to a dilution of 1:2,560. The preimmune IgY did not show any toxin B neutralizing activity at any of the concentrations tested. These results demonstrate that IgY purified from eggs laid by hens immunized simultaneously with toxin A and toxin B is an effective toxin B antitoxin.[0274]
In a separate study, the toxin B neutralizing activity of CTB, CTAB, and pre-immune IgY preparations was determined by reacting dilutions of these antibodies against a native toxin B concentration of 0.1 μg/ml (approximately 100% cytotoxic dose of toxin B in this assay system). The results are shown in FIG. 5.[0275]
Significant neutralization of toxin B occurred with the CTB IgY (antitoxin B, above) at dilutions of 1:80 and lower, while the CTAB IgY (antitoxin A+toxin B, above) was found to have significant neutralizing activity at dilutions of 1:40 and lower. The preimmune IgY did not show any toxin B neutralizing activity at any of the concentrations tested. These results demonstrate that IgY purified from eggs laid by hens immunized with toxin B alone, or simultaneously with toxin A and toxin B, is an effective toxin B antitoxin.[0276]

Example 9In vivo Protection of Golden Syrian Hamsters fromC. difficileDisease by Avian Antitoxins AgainstC. difficileToxins A and B

The most extensively used animal model to study[0277]C. difficiledisease is the hamster. [Lyerly et al., Infect. Immun. 47:349-352 (1992).] Several other animal models for antibiotic-induced diarrhea exist, but none mimic the human form of the disease as closely as the hamster model. [R. Fekety, “Animal Models of Antibiotic-Induced Colitis,” in O. Zak and M. Sande (eds.),Experimental Models in Antimicrobial Chemotherapy, Vol. 2, pp.61-72, (1986).] In this model, the animals are first predisposed to the disease by the oral administration of an antibiotic, such as clindamycin, which alters the population of normally-occurring gastrointestinal flora (Fekety, at 61-72). Following the oral challenge of these animals with viableC. difficileorganisms, the hamsters develop cecitis, and hemorrhage, ulceration, and inflammation are evident in the intestinal mucosa. [Lyerly et al.,Infect. Immun.47:349-352 (1985).] The animals become lethargic, develop severe diarrhea, and a high percentage of them die from the disease. [Lyerly et al.,Infect. Immun.47:349-352 (1985).] This model is therefore ideally suited for the evaluation of therapeutic agents designed for the treatment or prophylaxis ofC. difficiledisease.

The ability of the avian[0278]C. difficileantitoxins, described in Example 1 above, to protect hamsters fromC. difficiledisease was evaluated using the Golden Syrian hamster model ofC. difficileinfection. The Example involved (a) preparation of the avianC. difficileantitoxins, (b) in vivo protection of hamsters fromC. difficiledisease by treatment with avian antitoxins, and (c) long-term survival of treated hamsters.

a) Preparation of the Avian[0279]C. difficileAntitoxins

Eggs were collected from hens in groups CTA and CTAB described in Example 1 (b) above. To be used as a pre-immune (negative) control, eggs were also purchased from a local supermarket. Egg yolk immunoglobulin (IgY) was extracted from the 3 groups of eggs as described in Example 1 (c), and the final IgY pellets were solubilized in one fourth the original yolk volume of Ensure® nutritional formula.[0280]

b) In Vivo Protection of Hamsters Against[0281]C. difficileDisease by Treatment with Avian Antitoxins

The avian[0282]C. difficileantitoxins prepared in section (a) above were evaluated for their ability to protect hamsters fromC. difficiledisease using an animal model system which was modified from published procedures. [Fekety, at 61-72; Borriello et al., J. Med. Microbiol., 24:53-64 (1987); Kim et al., Infect. Immun., 55:2984-2992 (1987); Borriello et al., J. Med. Microbiol., 25:191-196 (1988); Delmee and Avesani, J. Med. Microbiol., 33:85-90 (1990); and Lyerly et al., Infect. Immun. 59:2215-2218 (1991).] For the study, three separate experimental groups were used, with each group consisting of 7 female Golden Syrian hamsters (Charles River), approximately 10 weeks old and weighing approximately 100 gms. each. The three groups were designated “CTA,” “CTAB” and “Pre-immune.” These designations corresponded to the antitoxin preparations with which the animals in each group were treated. Each animal was housed in an individual cage, and was offered food and water ad libitum through the entire length of the study. Onday 1, each animal was orally administered 1.0 ml of one of the three antitoxin preparations (prepared in section (a) above) at the following timepoints: 0 hrs., 4 hrs., and 8 hrs. Onday 2, theday 1 treatment was repeated. Onday 3, at the 0 hr. timepoint, each animal was again administered antitoxin, as described above. At 1 hr., each animal was orally administered 3.0 mg of clindamycin-HCl (Sigma) in 1 ml of water. This treatment predisposed the animals to infection withC. difficile. As a control for possible endogenousC. difficilecolonization, an additional animal from the same shipment (untreated) was also administered 3.0 mg of clindamycin-HCl in the same manner. This clindamycin control animal was left untreated (and uninfected) for the remainder of the study. At the 4 hr. and 8 hr. timepoints, the animals were administered antitoxin as described above. Onday 4, at the 0 hr. timepoint, each animal was again administered antitoxin as described above. At 1 hr., each animal was orally challenged with 1 ml ofC. difficileinoculum, which contained approx. 100C. difficilestrain 43596 organisms in sterile saline.C. difficilestrain 43596, which is a serogroup C strain, was chosen because it is representative of one of the most frequently-occurring serogroups isolated from patients with antibiotic-associated pseudomembranous colitis. [Delmee et al, J. Clin. Microbiol., 28:2210-2214 (1985).] In addition, this strain has been previously demonstrated to be virulent in the hamster model of infection. [Delmee and Avesani, J. Med. Microbiol., 33:85-90 (1990).] At the 4 hr. and 8 hr. timepoints, the animals were administered antitoxin as described above. Ondays 5 through 13, the animals were administeredantitoxin 3× per day as described forday 1 above, and observed for the onset of diarrhea and death. On the morning ofday 14, the final results of the study were tabulated. These results are shown in Table 13.

Representative animals from those that died in the Pre-Immune and CTA groups were necropsied. Viable[0283]C. difficileorganisms were cultured from the ceca of these animals, and the gross pathology of the gastrointestinal tracts of these animals was consistent with that expected forC. difficiledisease (inflamed, distended, hemorrhagic cecum, filled with watery diarrhea-like material). In addition, the clindamycin control animal remained healthy throughout the entire study period, therefore indicating that the hamsters used in the study had not previously been colonized with endogenousC. difficileorganisms prior to the start of the study. Following the final antitoxin treatment onday 13, a single surviving animal from the CTA group, and also from the CTAB group, was sacrificed and necropsied. No pathology was noted in either animal.

TABLE 13


Treatment Results

	No. Animals	No. Animals
Treatment Group	Surviving	Dead

Pre-Immune
	1	6
CTA (Antitoxin A only)	5	2
CTAB (Antitoxin A + Antitoxin B)	7	0

Treatment of hamsters with orally-administered toxin A and toxin B antitoxin (group CTAB) successfully protected 7 out of 7 (100%) of the animals from[0284]C. difficiledisease. Treatment of hamsters with orally-administered toxin A antitoxin (group CTA) protected 5 out of 7 (71%) of these animals fromC. difficiledisease. Treatment using pre-immune IgY was not protective againstC. difficiledisease, as only 1 out of 7 (14%) of these animals survived. These results demonstrate that the avian toxin A antitoxin and the avian toxin A+toxin B antitoxin effectively protected the hamsters fromC. difficiledisease. These results also suggest that although the neutralization of toxin A alone confers some degree of protection againstC. difficiledisease, in order to achieve maximal protection, simultaneous antitoxin A and antitoxin B activity is necessary.

c) Long-Term Survival of Treated Hamsters[0285]

It has been previously reported in the literature that hamsters treated with orally-administered bovine antitoxin IgG concentrate are protected from[0286]C. difficiledisease as long as the treatment is continued, but when the treatment is stopped, the animals develop diarrhea and subsequently die within 72 hrs. [Lyerly et al., Infect. Immun., 59(6):2215-2218 (1991).]

In order to determine whether treatment of[0287]C. difficiledisease using avian antitoxins promotes long-term survival following the discontinuation of treatment, the 4 surviving animals in group CTA, and the 6 surviving animals in group CTAB were observed for a period of 11 days (264 hrs.) following the discontinuation of antitoxin treatment described in section (b) above. All hamsters remained healthy through the entire post-treatment period. This result demonstrates that not only does treatment with avian antitoxin protect against the onset ofC. difficiledisease (i.e., it is effective as a prophylactic), it also promotes long-term survival beyond the treatment period, and thus provides a lasting cure.

Example 10In Vivo Treatment of EstablishedC. difficileInfection in Golden Syrian Hamsters with Avian Antitoxins AgainstC. difficileToxins A and B

The ability of the avian[0288]C. difficileantitoxins, described in Example 8 above, to treat an establishedC. difficile infection was evaluated using the Golden Syrian hamster model. The Example involved (a) preparation of the avianC. difficileantitoxins, (b) in vivo treatment of hamsters with establishedC. difficileinfection, and (c) histologic evaluation of cecal tissue.

a) Preparation Of The Avian[0289]C. difficileAntitoxins

Eggs were collected from hens in group CTAB described in Example 8 (b) above, which were immunized with[0290]C. difficiletoxoids and native toxins A and B. Eggs purchased from a local supermarket were used as a pre-immune (negative) control. Egg yolk immunoglobulin (IgY) was extracted from the 2 groups of eggs as described in Example 1 (c), and the final IgY pellets were solubilized in one-fourth the original yolk volume of Ensure® nutritional formula.

b) In Vivo Treatment of Hamsters with Established[0291]C. difficileInfection

The avian[0292]C. difficileantitoxins prepared in section (a) above were evaluated for the ability to treat establishedC. difficileinfection in hamsters using an animal model system which was modified from the procedure which was described for the hamster protection study in Example 8(b) above.

For the study, four separate experimental groups were used, with each group consisting of 7 female Golden Syrian hamsters (Charles River), approx. 10 weeks old, weighing approximately 100 gms. each. Each animal was housed separately, and was offered food and water ad libitum through the entire length of the study.[0293]

On[0294]day 1 of the study, the animals in all four groups were each predisposed toC. difficileinfection by the oral administration of 3.0 mg of clindamycin-HCl (Sigma) in 1 ml of water.

On[0295]day 2, each animal in all four groups was orally challenged with 1 ml ofC. difficileinoculum, which contained approximately 100C. difficilestrain 43596 organisms in sterile saline.C. difficilestrain 43596 was chosen because it is representative of one of the most frequently-occurring serogroups isolated from patients with antibiotic-associated pseudomembranous colitis. [Delmee et al., J. Clin. Microbiol., 28:2210-2214 (1990).] In addition, as this was the sameC. difficilestrain used in all of the previous Examples above, it was again used in order to provide experimental continuity.

On[0296]day 3 of the study (24 hrs. post-infection), treatment was started for two of the four groups of animals. Each animal of one group was orally administered 1.0 ml of the CTAB IgY preparation (prepared in section (a) above) at the following timepoints: 0 hrs., 4 hrs., and 8 hrs. The animals in this group were designated “CTAB-24.” The animals in the second group were each orally administered 1.0 ml of the pre-immune IgY preparation (also prepared in section (a) above) at the same timepoints as for the CTAB group. These animals were designated “Pre-24.” Nothing was done to the remaining two groups of animals onday 3.

On[0297]day 4, 48 hrs. post-infection, the treatment described forday 3 above was repeated for the CTAB-24 and Pre-24 groups, and was initiated for the remaining two groups at the same timepoints. The final two groups of animals were designated “CTAB-48” and “Pre-18” respectively.

On[0298]days 5 through 9, the animals in all four groups were administered antitoxin or pre-immune IgY, 3× per day, as described forday 4 above. The four experimental groups are summarized in Table 14.

TABLE 14


Experimental Treatment Groups

Group
Designation	Experimental Treatment

CTAB-24	Infected, treatment w/antitoxin IgY started @ 24 hrs.
	post-infection.
Pre-24	Infected, treatment w/pre-immune IgY started @ 24 hrs.
	post-infection.
CTAB-48	Infected, treatment w/antitoxin IgY started @ 48 hrs.
	post-infection.
Pre-48	Infected, treatment w/pre-immune IgY started @ 48 hrs.
	post-infection.

All animals were observed for the onset of diarrhea and death through the conclusion of the study on the morning of[0299]day 10. The results of this study are displayed in Table 15.

TABLE 15


Experimental Outcome -Day 10

Treatment Group	No. Animals Surviving	No. Animals Dead

CTAB-24	6	1
Pre-24	0	7
CTAB-48	4	3
Pre-48	2	5

Eighty-six percent of the animals which began receiving treatment with antitoxin IgY at 24 hrs. post-infection (CTAB-24 above) survived, while 57% of the animals treated with antitoxin IgY starting 48 hrs. post-infection (CTAB-48 above) survived. In contrast, none of the animals receiving pre-immune IgY starting 24 hrs. post-infection (Pre-24 above) survived, and only 29% of the animals which began receiving treatment with pre-immune IgY at 48 hrs. post-infection (Pre-48 above) survived through the conclusion of the study. These results demonstrate that avian antitoxins raised against[0300]C. difficiletoxins A and B are capable of successfully treating establishedC. difficileinfections in vivo.

c) Histologic Evaluation of Cecal Tissue[0301]

In order to further evaluate the ability of the IgY preparations tested in this study to treat established[0302]C. difficileinfection, histologic evaluations were performed on cecal tissue specimens obtained from representative animals from the study described in section (b) above.

Immediately following death, cecal tissue specimens were removed from animals which died in the Pre-24 and Pre-48 groups. Following the completion of the study, a representative surviving animal was sacrificed and cecal tissue specimens were removed from the CTAB-24 and CTAB-48 groups. A single untreated animal from the same shipment as those used in the study was also sacrificed and a cecal tissue specimen was removed as a normal control. All tissue specimens were fixed overnight at 4° C. in 10% buffered formalin. The fixed tissues were paraffin-embedded, sectioned, and mounted on glass microscope slides. The tissue sections were then stained using hematoxylin and eosin (H and E stain), and were examined by light microscopy.[0303]

Upon examination, the tissues obtained from the CTAB-24 and CTAB-48 animals showed no pathology, and were indistinguishable from the normal control. This observation provides further evidence for the ability of avian antitoxins raised against[0304]C. difficiletoxins A and B to effectively treat establishedC. difficileinfection, and to prevent the pathologic consequences which normally occur as a result ofC. difficiledisease.

In contrast, characteristic substantial mucosal damage and destruction was observed in the tissues of the animals from the Pre-24 and Pre-48 groups which died from[0305]C. difficiledisease. Normal tissue architecture was obliterated in these two preparations, as most of the mucosal layer was observed to have sloughed away, and there were numerous large hemorrhagic areas containing massive numbers of erythrocytes.

Example 11Cloning and Expression ofC. difficileToxin A Fragments

The toxin A gene has been cloned and sequenced, and shown to encode a protein of predicted MW of 308 kd. [Dove et al., Infect. Immun., 58:480-488 (1990).] Given the expense and difficulty of isolating native toxin A protein, it would be advantageous to use simple and inexpensive procaryotic expression systems to produce and purify high levels of recombinant toxin A protein for immunization purposes. Ideally, the isolated recombinant protein would be soluble in order to preserve native antigenicity, since solubilized inclusion body proteins often do not fold into native conformations. To allow ease of purification, the recombinant protein should be expressed to levels greater than 1 mg/liter of[0306]E. coliculture.

To determine whether high levels of recombinant toxin A protein can be produced in[0307]E. coli, fragments of the toxin A gene were cloned into various prokaryotic expression vectors, and assessed for the ability to express recombinant toxin A protein inE. coli. Three prokaryotic expression systems were utilized. These systems were chosen because they drive expression of either fusion (pMALc and pGEX2T) or native (pET23a-c) protein to high levels inE. coli, and allow affinity purification of the expressed protein on a ligand containing column. Fusion proteins expressed from pGEX vectors bind glutathione agarose beads, and are eluted with reduced glutathione. pMAL fusion proteins bind amylose resin, and are eluted with maltose. A poly-histidine tag is present at either the N-terminal (pET16b) or C-terminal (pET23a-c) end of pET fusion proteins. This sequence specifically binds Ni₂⁺ chelate columns, and is eluted with imidazole salts. Extensive descriptions of these vectors are available [Williams et al. (1995)DNA Cloning2: Expression Systems, Glover and Hames, eds. IRL Press, Oxford, pp. 15-58], and will not be discussed in detail here. The Example involved (a) cloning of the toxin A gene, (b) expression of large fragments of toxin A in various prokaryotic expression systems, (c) identification of smaller toxin A gene fragments that express efficiently inE. coli, (d) purification of recombinant toxin A protein by affinity chromatography, and (e) demonstration of functional activity of a recombinant fragment of the toxin A gene.

a) Cloning of the Toxin A Gene[0308]

A restriction map of the toxin A gene is shown in FIG. 6. The encoded protein contains a carboxy terminal ligand binding region, containing multiple repeats of a carbohydrate binding domain. [von Eichel-Streiber and Sauerbom, Gene 96:107-113 (1990).] The toxin A gene was cloned in three pieces, by using either the polymerase chain reaction (PCR) to amplify specific regions, ([0309]

regions

1 and 2, FIG. 6) or by screening a constructed genomic library for a specific toxin A gene fragment (region 3, FIG. 6). The sequences of the utilized PCR primers are P1: 5′GGAAATT TAGCTGCAGCATCTGAC 3′ (SEQ ID NO.: 1); P2: 5′TCTAGCAAATTCGCTTGT GTTGAA 3′ (SEQ ID NO.:2); P3: 5′CTCGCATATAGCATTAGACC 3′ (SEQ ID NO.:3); and P4: 5′CTATCTAGGCCTAAAGTAT 3′ (SEQ ID NO.:4). These regions were cloned into prokaryotic expression vectors that express either fusion (pMALc and pGEX2T) or native (pET23a-c) protein to high levels inE. coli, and allow affinity purification of the expressed protein on a ligand containing column.

[0310]Clostridium difficileVPI strain 10463 was obtained from the ATCC (ATCC #43255) and grown under anaerobic conditions in brain-heart infusion medium (BBL). High molecular-weightC. difficileDNA was isolated essentially as described by Wren and Tabaqchali (1987) J. Clin. Microbiol., 25:2402, except proteinase K and sodium dodecyl sulfate (SDS) was used to disrupt the bacteria, and cetyltrimethylammonium bromide precipitation [as described in Ausubel et al.,Current Protocols in Molecular Biology(1989)] was used to remove carbohydrates from the cleared lysate. The integrity and yield of genomic DNA was assessed by comparison with a serial dilution of uncut lambda DNA after electrophoresis on an agarose gel.

Fragments 1 and 2 were cloned by PCR, utilizing a proofreading thermostable DNA polymerase (native pfu polymerase; Stratagene). The high fidelity of this polymerase reduces the mutation problems associated with amplification by error prone polymerases (e.g., Taq polymerase). PCR amplification was performed using the indicated PCR primers (FIG. 6) in 50 pI reactions containing 10 mM Tris-HCl(8.3), 50 mM KCl, 1.5 mM MgCl[0311]₂, 200 μM each dNTP, 0.2 μM each primer, and 50 ngC. difficilegenomic DNA. Reactions were overlaid with 100 μl mineral oil, heated to 94° C. for 4 min, 0.5 μl native pfu polymerase (Stratagene) added, and the reaction cycled 30× at 94° C. for 1 min, 50° C. for 1 min, 72° C. for 4 min, followed by 10 min at 72° C. Duplicate reactions were pooled, chloroform extracted, and ethanol precipitated. After washing in 70% ethanol, the pellets were resuspended in 50 μl TE buffer [10 mM Tris-HCL, 1 mM EDTA pH 8.0]. Aliquots of 10 μl each were restriction digested with either EcoRI/HincII (fragment 1) or EcoRI/PstI (fragment 2), and the appropriate restriction fragments were gel purified using the Prep-A-Gene kit (BioRad), and ligated to either EcoRI/SmaI-restricted pGEX2T (Pharmacia) vector (fragment 1), or the EcoRI/PstI pMAlc (New England Biolabs) vector (fragment 2). Both clones are predicted to produce in-frame fusions with either the glutathione-S-transferase protein (pGEX vector) or the maltose binding protein (pMAL vector). Recombinant clones were isolated, and confirmed by restriction digestion, using standard recombinant molecular biology techniques. [Sambrook et al.,Molecular Cloning, A Laboratory Manual(1989), and designated pGA30-660 and pMA660-1100, respectively (see FIG. 6 for description of the clone designations).]

[0312]Fragment 3 was cloned from a genomic library of size selected PstI digestedC. difficilegenomic DNA, using standard molecular biology techniques (Sambrook et al.). Given that thefragment 3 internal PstI site is protected from cleavage inC. difficilegenomic DNA [Price et al., Curr. Microbiol., 16:55-60 (1987)], a 4.7 kb fragment from PstI restrictedC. difficilegenomic DNA was gel purified, and ligated to PstI restricted, phosphatase treated pUC9 DNA. The resulting genomic library was screened with a oligonucleotide primer specific tofragment 3, and multiple independent clones were isolated. The presence offragment 3 in several of these clones was confirmed by restriction digestion, and a clone of the indicated orientation (FIG. 6) was restricted with BamHI/HindIII, the released fragment purified by gel electrophoresis, and ligated into similarly restricted pET23c expression vector DNA (Novagen). Recombinant clones were isolated, and confirmed by restriction digestion. This construct is predicted to create both a predicted in frame fusion with the pET protein leader sequence, as well as a predicted C-terminal poly-histidine affinity tag, and is designated pPA1100-2680 (see FIG. 6 for the clone designation).

b) Expression of Large Fragments of Toxin A in[0313]E. coli

Protein expression from the three expression constructs made in (a) was induced, and analyzed by Western blot analysis with an affinity purified, goat polyclonal antiserum directed against the toxin A toxoid (Tech Lab). The procedures utilized for protein induction, SDS-PAGE, and Western blot analysis are described in detail in Williams et al (1995), supra. In brief, 5[0314]ml 2×YT (16 g tryptone, 10 g yeast extract, 5 g NaCl per liter, pH 7.5+100 μg/ml ampicillin were added to cultures of bacteria (BL21 for pMAl and pGEX plasmids, and BL21(DE3)LysS for pET plasmids) containing the appropriate recombinant clone which were induced to express recombinant protein by addition of IPTG to 1 mM. Cultures were grown at 37° C., and induced when the cell density reached 0.5 OD₆₀₀. Induced protein was allowed to accumulate for two hrs after induction. Protein samples were prepared by pelleting 1 ml aliquots of bacteria by centrifugation (1 min in a microfuge), and resuspension of the pelleted bacteria in 150 μl of 2×SDS-PAGE sample buffer [Williams et al. (1995), supra]. The samples were heated to 95° C. for 5 min, the cooled and 5 or 10 μl aliquots loaded on 7.5% SDS-PAGE gels. BioRad high molecular weight protein markers were also loaded, to allow estimation of the MW of identified fusion proteins. After electrophoresis, protein was detected either generally by staining gels with Coomassie blue, or specifically, by blotting to nitrocellulose for Western blot detection of specific immunoreactive protein. Western blots, (performed as described in Example 3) which detect toxin A reactive protein in cell lysates of induced protein from the three expression constructs are shown in FIG. 7. In this figure, lanes 1-3 contain cell lysates prepared fromE. colistrains containing pPA1100-2860 in B 121 (DE3)lysE cells; lanes 4-6 contain cell lysates prepared fromE. colistrains containing pPA1100-2860 in B121(DE3)lysS cells; lanes 7-9 contain cell lysates prepared fromE. colistrains containing pMA30-660; lanes 10-12 contain cell lysates prepared fromE. colistrains containing pMA660-1100. The lanes were probed with an affinity purified goat antitoxin A polyclonal antibody (Tech Lab). Control lysates from uninduced cells. (

lanes

1, 7, and 10) contain very little immunoreactive material compared to the induced samples in the remaining lanes. The highest molecular weight band observed for each clone is consistent with the predicted size of the full length fusion protein.

Each construct directs expression of high molecular weight (HMW) protein that is reactive with the toxin A antibody. The size of the largest immunoreactive bands from each sample is consistent with predictions of the estimated MW of the intact fusion proteins. This demonstrates that the three fusions are in-frame, and that none of the clones contain cloning artifacts that disrupt the integrity of the encoded fusion protein. However, the Western blot demonstrates that fusion protein from the two larger constructs (pGA30-660 and pPA1100-2680) are highly degraded. Also, expression levels of toxin A proteins from these two constructs are low, since induced protein bands are not visible by Coomassie staining (not shown). Several other expression constructs that fuse large sub-regions of the toxin A gene to either pMALc or pET23a-c expression vectors, were constructed and tested for protein induction. These constructs were made by mixing gel purified restriction fragments, derived from the expression constructs shown in FIG. 6, with appropriately cleaved expression vectors, ligating, and selecting recombinant clones in which the toxin A restriction fragments had ligated together and into the expression vector as predicted for in-frame fusions. The expressed toxin A interval within these constructs are shown in FIG. 8, as well as the internal restriction sites utilized to make these constructs.[0315]

As used herein, the term “interval” refers to any portion (i.e., any segment of the toxin which is less than the whole toxin molecule) of a clostridial toxin. In a preferred embodiment, “interval” refers to portions of[0316]C. difficiletoxins such as toxin A or toxin B. It is also contemplated that these intervals will correspond to epitopes of immunologic importance, such as antigens or immunogens against which a neutralizing antibody response is effected. It is not intended that the present invention be limited to the particular intervals or sequences described in these Examples. It is also contemplated that sub-portions of intervals (e.g., an epitope contained within one interval or which bridges multiple intervals) be used as compositions and in the methods of the present invention.

In all cases, Western blot analysis of each of these constructs with goat antitoxin A antibody (Tech Lab) detected HMW fusion protein of the predicted size (not shown). This confirms that the reading frame of each of these clones is not prematurely terminated, and is fused in the correct frame with the fusion partner. However, the Western blot analysis revealed that in all cases, the induced protein is highly degraded, and, as assessed by the absence of identifiable induced protein bands by Coomassie Blue staining, are expressed only at low levels. These results suggest that expression of high levels of intact toxin A recombinant protein is not possible when large regions of the toxin A gene are expressed in[0317]E. coliusing these expression vectors.

c) High Level Expression of Small Toxin A Protein Fusions in[0318]E. coli

Experience indicates that expression difficulties are often encountered when large (greater than 100 kd) fragments are expressed in[0319]E coli. A number of expression constructs containing smaller fragments of the toxin A gene were constructed, to determine if small regions of the gene can be expressed to high levels without extensive protein degradation. A summary of these expression constructs are shown in FIG. 9. All were constructed by in-frame fusions of convenient toxin A restriction fragments to either the pMALc or pET23a-c vectors. Protein preparations from induced cultures of each of these constructs were analyzed by both Coomassie Blue staining and Western analysis as in (b) above. In all cases, higher levels of intact, full length fusion proteins were observed than with the larger recombinants from section (b).

d) Purification of Recombinant Toxin A Protein Large scale (500 ml) cultures of each recombinant from (c) were grown, induced, and soluble and insoluble protein fractions were isolated. The soluble protein extracts were affinity chromatographed to isolate recombinant fusion protein, as described [Williams et al. (1994), supra]. In brief, extracts containing tagged pET fusions were chromatographed on a nickel chelate column, and eluted using imidazole salts as described by the distributor (Novagen). Extracts containing soluble pMAL fusion protein were prepared and chromatographed in column buffer (10 mM NaPO[0320]₄, 0.5M NaCl, 10 mM β-mercaptoethanol, pH 7.2) over an amylose resin column (New England Biolabs), and eluted with column buffer containing 10 mM maltose as described [Williams et al. (1995), supra]. When the expressed protein was found to be predominantly insoluble, insoluble protein extracts were prepared by the method described in Example 17, infra. The results are summarized in Table 16. FIG. 10 shows the sample purifications of recombinant toxin A protein. In this figure,

lanes

1 and 2 contain MBP fusion protein purified by affinity purification of soluble protein.

TABLE 16


Purification Of Recombinant Toxin A Protein

		Yield Affinity		Yield Intact
	Protein	Purified Soluble	% Intact Soluble	Insoluble Fusion
Clone^(a)	Solubility	Protein^(b)	Fusion Protein^(c)	Protein

pMA30-270	Soluble	4	mg/500mls	10%	NA
PMA30-300	Soluble	4	mg/500 mls	5-10%	NA

pMA660-1100	Soluble	4.5	mg/500mls	50%	NA
pMA1100-1610	Soluble	18	mg/500mls	10%	NA
pMA1610-1870	Both	22	mg/500mls	90%	20 mg/500 ml

pPA1100-1450	Soluble	0.1	mg/500mls	90%	NA
pPA1100-1870	Soluble	0.02	mg/500mls	90%	NA
pMA1870-2680	Both	12	mg/500mls	80%	NA

pPa1870-2680	Insoluble	—	NA	10 mg/500 ml

[0321]

Lanes

3 and 4 contain MBP fusion protein purified by solubilization of insoluble inclusion bodies. The purified fusion protein sarnples are pMA1870-2680 (lane 1), pMA660-1100 (lane 2), pMA300-600 (lane 3) and pMA1450-1870 (lane 4).

Poor yields of affinity purified protein were obtained when poly-histidine tagged pET vectors were used to drive expression (pPA1100-1450, pP1100-1870). However, significant protein yields were obtained from pMAL expression constructs spanning the entire toxin A gene, and yields of full-length soluble fusion protein ranged from an estimated 200-400 μg/500 ml culture (pMA30-300) to greater than 20 mg/500 ml culture (pMA1610-1870). Only one interval was expressed to high levels as strictly insoluble protein (pMA300-660). Thus, although high level expression was not observed when using large expression constructs from the toxin A gene, usable levels of recombinant protein spanning the entire toxin A gene were obtainable by isolating induced protein from a series of smaller pMAL expression constructs that span the entire toxin A gene. This is the first demonstration of the feasibility of expressing recombinant toxin A protein to high levels in[0322]E. coli.

e) Hemagglutination Assay Using the Toxin A Recombinant Proteins[0323]

The carboxy terminal end consisting of the repeating units contains the hemagglutination activity or binding domain of[0324]C. difficiletoxin A. To determine whether the expressed toxin A recombinants retain functional activity, hemagglutination assays were performed. Two toxin A recombinant proteins, one containing the binding domain as either soluble affinity purified protein (pMA1870-2680) or SDS solubilized inclusion body protein (pPA1870-2680) and soluble protein from one region outside that domain (pMA1100-1610) were tested using a described procedure. [H. C. Krivan et. al., Infect. Immun., 53:573 (1986).] Citrated rabbit red blood cells (RRBC)(Cocalico) were washed several times with Tris-buffer (0.1M Tris and 50 mM NaCl) by centrifugation at 450×g for 10 minutes at 4° C. A 1% RRBC suspension was made from the packed cells and resuspended in Tris-buffer. Dilutions of the recombinant proteins and native toxin A (Tech Labs) were made in the Tris-buffer and added in duplicate to a round-bottomed 96-well microtiter plate in a final volume of 100 μl. To each well, 50 μl of the 1% RRBC suspension was added, mixed by gentle tapping, and incubated at 4° C. for 3-4 hours. Significant hemagglutination occurred only in the recombinant proteins containing the binding domain (pMA 1870-2680) and native toxin A. The recombinant protein outside the binding domain (pMA 1100-1610) displayed no hemagglutination activity. Using equivalent protein concentrations, the hemagglutination titer for toxin A was 1:256, while titers for the soluble and insoluble recombinant proteins of the binding domain were 1:256 and about 1:5000. Clearly, the recombinant proteins tested retained functional activity and were able to bind RRBC's.

Example 12Functional Activity of IgY Reactive Against Toxin A Recombinants

The expression of recombinant toxin A protein as multiple fragments in[0325]E. colihas demonstrated the feasibility of generating toxin A antigen through use of recombinant methodologies (Example 11). The isolation of these recombinant proteins allows the irmunoreactivity of each individual subregion of the toxin A protein to be determined (i.e., in a antibody pool directed against the native toxin A protein). This identifies the regions (if any) for which little or no antibody response is elicited when the whole protein is used as a immunogen. Antibodies directed against specific fragments of the toxin A protein can be purified by affinity chromatography against recombinant toxin A protein, and tested for neutralization ability. This identifies any toxin A subregions that are essential for producing neutralizing antibodies. Comparison with the levels of immune response directed against these intervals when native toxin is used as an immunogen predicts whether potentially higher titers of neutralizing antibodies can be produced by using recombinant protein directed against a individual region, rather than the entire protein. Finally, since it is unknown whether antibodies reactive to the recombinant toxin A proteins produced in Example 11 neutralize toxin A as effectively as antibodies raised against native toxin A (Examples 9 and 10), the protective ability of a pool of antibodies affinity purified against recombinant toxin A fragments was assessed for its ability to neutralize toxin A.

This Example involved (a) epitope mapping of the toxin A protein to determine the titre of specific antibodies directed against individual subregions of the toxin A protein when native toxin A protein is used as an immunogen, (b) affinity purification of IgY reactive against recombinant proteins spanning the toxin A gene, (c) toxin A neutralization assays with affinity purified IgY reactive to recombinant toxin A protein to identify subregions of the toxin A protein that induce the production of neutralizing antibodies, and determination of whether complete neutralization of toxin A can be elicited with a mixture of antibodies reactive to recombinant toxin A protein.[0326]

a) Epitope Mapping of the Toxin A Gene[0327]

The affinity purification of recombinant toxin A protein specific to defined intervals of the toxin A protein allows epitope mapping of antibody pools directed against native toxin A. This has not previously been possible, since previous expression of toxin A recombinants has been assessed only by Western blot analysis, without knowledge of the expression levels of the protein [e.g., von Eichel-Streiber et al, J. Gen. Microbiol., 135:55-64 (1989)]. Thus, high or low reactivity of recombinant toxin A protein on Western blots may reflect protein expression level differences, not immunoreactivity differences. Given that the purified recombinant protein generated in Example 11 have been quantitated, the issue of relative immunoreactivity of individual regions of the toxin A protein was precisely addressed.[0328]

For the purposes of this Example, the toxin A protein was subdivided into 6 intervals (1-6), numbered from the amino (interval 1) to the carboxyl (interval 6) termini.[0329]

The recombinant proteins corresponding to these intervals were from expression clones (see Example 11(d) for clone designations) pMA30-300 (interval 1), pMA300-660 (interval 2), pMA660-1100 (interval 3), pPA1100-1450 (interval 4), pMA1450-1870 (interval 5) and pMA1870-2680 (interval 6). These 6 clones were selected because they span the entire protein from amino acids numbered 30 through 2680, and subdivide the protein into 6 small intervals. Also, the carbohydrate binding repeat interval is contained specifically in one interval (interval 6), allowing evaluation of the immune response specifically directed against this region. Western blots of 7.5% SDS-PAGE gels, loaded and electrophoresed with defined quantities of each recombinant protein, were probed with either goat antitoxin A polyclonal antibody (Tech Lab) or chicken antitoxin A polyclonal antibody [pCTA IgY, Example 8(c)]. The blots were prepared and developed with alkaline phosphatase as previously described [Williams et al. (1995), supra]. At least 90% of all reactivity, in either goat or chicken antibody pools, was found to be directed against the ligand binding domain (interval 6). The remaining immunoreactivity was directed against all five remaining intervals, and was similar in both antibody pools, except that the chicken antibody showed a much lower reactivity against[0330]interval 2 than the goat antibody.

This clearly demonstrates that when native toxin A is used as an immunogen in goats or chickens, the bulk of the immune response is directed against the ligand binding domain of the protein, with the remaining response distributed throughout the remaining ⅔ of the protein.[0331]

b) Affinity Purification of IgY Reactive Against Recombinant Toxin A Protein[0332]

Affinity columns, containing recombinant toxin A protein from the 6 defined intervals in (a) above, were made and used to (i) affinity purify antibodies reactive to each individual interval from the CTA IgY preparation [Example 8(c)], and (ii) deplete interval specific antibodies from the CTA IgY preparation. Affinity columns were made by coupling 1 ml of PBS-washed Actigel resin (Sterogene) with region specific protein and {fraction (1/10)} final volume of Ald-coupling solution (1M sodium cyanoborohydride). The total region specific protein added to each reaction mixture was 2.7 mg (interval 1), 3 mg ([0333]intervals 2 and 3), 0.1 mg (interval 4), 0.2 mg (interval 5) and 4 mg (interval 6). Protein for

intervals

1, 3, and 6 was affinity purified pMAl fusion protein in column buffer (see Example 11).Interval 4 was affinity purified poly-histidine containing pET fusion in PBS;

intervals

2 and 5 were from inclusion body preparations of insoluble pMAL fusion protein, dialyzed extensively in PBS. Aliquots of the supernatants from the coupling reactions, before and after coupling, were assessed by Coomassie staining of 7.5% SDS-PAGE gels. Based on protein band intensities, in all cases greater than 50% coupling efficiencies were estimated. The resins were poured into 5 ml BioRad columns, washed extensively with PBS, and stored at 4° C.

Aliquots of the CTA IgY polyclonal antibody preparation were depleted for each individual region as described below. A 20 ml sample of the CTA IgY preparation [Example 8(c)] was dialyzed extensively against 3 changes of PBS (1 liter for each dialysis), quantitated by absorbance at OD[0334]₂₈₀, and stored at 4° C. Six 1 ml aliquots of the dialyzed IgY preparation were removed, and depleted individually for each of the six intervals. Each 1 ml aliquot was passed over the appropriate affinity column, and the eluate twice reapplied to the column. The eluate was collected, and pooled with a 1 ml PBS wash. Bound antibody was eluted from the column by washing with 5 column volumes of 4 M Guanidine-HCl (in 10 mM Tris-HCl, pH 8.0). The column was reequilibrated in PBS, and the depleted antibody stock reapplied as described above. The eluate was collected, pooled with a 1 ml PBS wash, quantitated by absorbance at OD₂₈₀, and stored at 4° C. In this manner, 6 aliquots of the CTA IgY preparation were individually depleted for each of the 6 toxin A intervals, by two rounds of affinity depletion. The specificity of each depleted stock was tested by Western blot analysis. Multiple 7.5% SDS-PAGE gels were loaded with protein samples corresponding to all 6 toxin A subregions. After electrophoresis, the gels were blotted, and protein transfer confirmed by Ponceau S staining [protocols described in Williams et al. (1995), supra]. After blocking theblots 1 hr at 20° C. in PBS+0.1% Tween 20 (PBST) containing 5% milk (as a blocking buffer), 4 ml of either a 1/500 dilution of the dialyzed CTA IgY preparation in blocking buffer, or an equivalent arnount of the six depleted antibody stocks (using OD₂₈₀to standardize antibody concentration) were added and the blots incubated a further 1 hr at room temperature. The blots were washed and developed with alkaline phosphatase (using a rabbit anti-chicken alkaline phosphate conjugate as a secondary antibody) as previously described [Williams et al. (1995), supra]. In all cases, only the target interval was depleted for antibody reactivity, and at least 90% of the reactivity to the target intervals was specifically depleted.

Region specific antibody pools were isolated by affinity chromatography as described below. Ten mis of the dialyzed CTA IgY preparation were applied sequentially to each affinity column, such that a single 10 ml aliquot was used to isolate region specific antibodies specific to each of the six subregions. The columns were sequentially washed with 10 volumes of PBS, 6 volumes of BBS-Tween, 10 volumes of TBS, and eluted with 4 ml Actisep elution media (Sterogene). The eluate was dialyzed extensively against several changes of PBS, and the affinity purified antibody collected and stored at 4° C. The volumes of the eluate increased to greater than 10 mls during dialysis in each case, due to the high viscosity of the Actisep elution media. Aliquots of each sample were 20× concentrated using Centricon.30 microconcentrators (Amicon) and stored at 4° C. The specificity of each region specific antibody pool was tested, relative to the dialyzed CTA IgY preparation, by Western blot analysis, exactly as described above, except that 4 ml samples of blocking buffer containing 1001 μl region specific antibody (unconcentrated) were used instead of the depleted CTA IgY preparations. Each affinity purified antibody preparation was specific to the defined interval, except that samples purified against intervals 1-5 also reacted with[0335]interval 6. This may be due to non-specific binding to theinterval 6 protein, since this protein contains the repetitive ligand binding domain which has been shown to bind antibodies nonspecifically. [Lyerly et al., Curr. Microbiol., 19:303-306 (1989).]

The reactivity of each affinity purified antibody preparation to the corresponding proteins was approximately the same as the reactivity of the a 1/500 diluted dialyzed CTA IgY preparation standard. Given that the specific antibody stocks were diluted 1/40, this would indicate that the unconcentrated affinity purified antibody stocks contain 1/10-1/20 the concentration of specific antibodies relative to the starting CTA IgY preparation.[0336]

c) Toxin A Neutralization Assay Using Antibodies Reactive Toward Recombinant Toxin A Protein[0337]

The CHO toxin neutralization assay [Example 8(d)] was used to assess the ability of the depleted or enriched samples generated in (b) above to neutralize the cytotoxicity of toxin A. The general ability of affinity purified antibodies to neutralize toxin A was assessed by mixing together aliquots of all 6 concentrated stocks of the 6 affinity purified samples generated in (b) above, and testing the ability of this mixture to neutralize a toxin A concentration of 0.1 μg/ml. The results, shown in FIG. 11, demonstrate almost complete neutralization of toxin A using the affinity purified (AP) mix. Some epitopes within the recombinant proteins utilized for affinity purification were probably lost when the proteins were denatured before affinity purification [by Guanidine-HCl treatment in (b) above]. Thus, the neutralization ability of antibodies directed against recombinant protein is probably underestimated using these affinity purified antibody pools. This experiment demonstrates that antibodies reactive to recombinant toxin A can neutralize cytotoxicity, suggesting that neutralizing antibodies may be generated by using recombinant toxin A protein as immunogen.[0338]

In view of the observation that the recombinant expression clones of the toxin A gene divide the protein into 6 subregions, the neutralizing ability of antibodies directed against each individual region was assessed. The neutralizing ability of antibodies directed against the ligand binding domain of toxin A was determined first.[0339]

In the toxin neutralization experiment shown in FIG. 11,[0340]interval 6 specific antibodies (interval 6 contains the ligand binding domain) were depleted from the dialyzed PEG preparation, and the effect on toxin neutralization assayed.Interval 6 antibodies were depleted either by utilizing theinterval 6 depleted CTA IgY preparation from (b) above (“−6 aff. depleted” in FIG. 11), or by addition ofinterval 6 protein to the CTA IgY preparation (estimated to be a 10 fold molar excess over anti-interval 6 immunoglobulin present in this preparation) to competitively compete forinterval 6 protein (”-6 prot depleted” in FIG. 11). In both instances, removal ofinterval 6 specific antibodies reduces the neutralization efficiency relative to the starting CTA IgY preparation. This demonstrates that antibodies directed againstinterval 6 contribute to toxin neutralization. Sinceinterval 6 corresponds to the ligand binding domain of the protein, these results demonstrate that antibodies directed against this region in the PEG preparation contribute to the neutralization of toxin A in this assay. However, it is significant that after removal of these antibodies, the PEG preparation retains significant ability to neutralize toxin A (FIG. 11). This neutralization is probably due to the action of antibodies specific to other regions of the toxin A protein, since at least 90% of the ligand binding region reactive antibodies were removed in the depleted sample prepared in (b) above. This conclusion was supported by comparison of the toxin neutralization of the affinity purified (AP) mix compared to affinity purifiedinterval 6 antibody alone. Although some neutralization ability was observed withAP interval 6 antibodies alone, the neutralization was significantly less than that observed with the mixture of all 6 AP antibody stocks (not shown).

Given that the mix of all six affinity purified samples almost completely neutralized the cytotoxicity of toxin A (FIG. 11), the relative importance of antibodies directed against toxin A intervals 1-5 within the mixture was determined. This was assessed in two ways. First, samples containing affinity purified antibodies representing 5 of the 6 intervals were prepared, such that each individual region was depleted from one sample. FIG. 12 demonstrates a sample neutralization curve, comparing the neutralization ability of affinity purified antibody mixes without interval 4 (−4) or 5 (−5) specific antibodies, relative to the mix of all 6 affinity purified antibody stocks (positive control). While the removal of[0341]interval 5 specific antibodies had no effect on toxin neutralization (or intervals 1-3, not shown), the loss ofinterval 4 specific antibodies significantly reduced toxin neutralization (FIG. 12).

Example 13Production and Evaluation of Avian Antitoxin AgainstC. difficileRecombinant Toxin A Polypeptide

In Example 12, we demonstrated neutralization of toxin A mediated cytotoxicity by affinity purified antibodies reactive to recombinant toxin A protein. To determine whether antibodies raised against a recombinant polypeptide fragment of[0343]C. difficiletoxin A may be effective in treating clostridial diseases, antibodies to recombinant toxin A protein representing the binding domain were generated. Two toxin A binding domain recombinant polypeptides, expressing the binding domain in either the pMALc (pMA1870-2680) or pET 23(pPA1870-2680) vector, were used as immunogens. The pMAL protein was affinity purified as a soluble product [Example 12(d)] and the pET protein was isolated as insoluble inclusion bodies [Example 12(d)] and solubilized to an immunologically active protein using a proprietary method described in a pending patent application (U.S. patent application Ser. No. 08/129,027). This Example involves (a) immunization, (b) antitoxin collection, (c) determination of antitoxin antibody titer, (d) anti-recombinant toxin A neutralization of toxin A hemagglutination activity in vitro, and (e) assay of in vitro toxin A neutralizing activity.

a) Immunization[0344]

The soluble and the inclusion body preparations each were used separately to immunize hens. Both purified toxin A polypeptides were diluted in PBS and emulsified with approximately equal volumes of CFA for the initial immunization or IFA for subsequent booster immunizations. On day zero, for each of the recombinant preparations, two egg laying white Leghorn hens (obtained from local breeder) were each injected at multiple sites (intramuscular and subcutaneous) with 1 ml of recombinant adjuvant mixture containing approximately 0.5 to 1.5 mgs of recombinant toxin A. Booster immunizations of 1.0 mg were given on[0345]days 14 andday 28.

b) Antitoxin Collection[0346]

Total yolk immune IgY was extracted as described in the standard PEG protocol (as in Example 1) and the final IgY pellet was dissolved in sterile PBS at the original yolk volume. This material is designated “immune recombinant IgY” or “immune IgY.”[0347]

c) Antitoxin Antibody Titer[0348]

To determine if the recombinant toxin A protein was sufficiently imnunogenic to raise antibodies in hens, the antibody titer of a recombinant toxin A polypeptide was determined by ELISA. Eggs from both hens were collected on day 32, the yolks pooled and the antibody was isolated using PEG as described. The immune recombinant IgY antibody titer was determined for the soluble recombinant protein containing the maltose binding protein fusion generated in p-Mal (pMA1870-2680). Ninety-six well Falcon Pro-bind plates were coated overnight at 4° C. with 100 μl/well of toxin A recombinant at 2.5 μg/μl in PBS containing 0.05% thimerosal. Another plate was also coated with maltose binding protein (MBP) at the same concentration, to permit comparison of antibody reactivity to the fusion partner. The next day, the wells were blocked with PBS containing 1% bovine serum albumin (BSA) for 1 hour at 37° C. IgY isolated from immune or preimmune eggs was diluted in antibody diluent (PBS containing 1% BSA and 0.05% Tween-20), and added to the blocked wells and incubated for 1 hour at 37° C. The plates were washed three times with PBS with 0.05% Tween-20, then three times with PBS. Alkaline phosphatase conjugated rabbit anti-chicken IgG (Sigma) diluted 1:1000 in antibody diluent was added to the plate, and incubated for 1 hour at 37° C. The plates were washed as before and substrate was added, [p-nitrophenyl phosphate (Sigma)] at 1 mg/ml in 0.05M Na[0349]₂CO₃, pH 9.5 and 10 mM MgCl₂. The plates were evaluated quantitatively on a Dynatech MR 300 Micro EPA plate reader at 410 nm about 10 minutes after the addition of substrate.

Based on these ELISA results, high antibody titers were raised in chickens immunized with the toxin A recombinant polypeptide. The recombinant appeared to be highly immunogenic, as it was able to generate high antibody titers relatively quickly with few immunizations. Immune IgY titer directed specifically to the toxin A portion of the recombinant was higher than the immune IgY titer to its fusion partner, the maltose binding protein, and significantly higher than the preimmune IgY. ELISA titers (reciprocal of the highest dilution of IgY generating a signal) in the preimmune IgY to the MBP or the recombinant was <1:30 while the immune IgY titers to MBP and the toxin A recombinant were 1:18750 and >1:93750 respectively. Importantly, the anti-recombinant antibody titers generated in the hens against the recombinant polypeptide is much higher, compared to antibodies to that region raised using native toxin A. The recombinant antibody titer to region 1870-2680 in the CTA antibody preparation is at least five-fold lower compared to the recombinant generated antibodies (1:18750 versus >1:93750). Thus, it appears a better immune response can be generated against a specific recombinant using that recombinant as the immunogen compared to the native toxin A.[0350]

This observation is significant, as it shows that because recombinant portions stimulate the production of antibodies, it is not necessary to use native toxin molecules to produce antitoxin preparations. Thus, the problems associated with the toxicity of the native toxin are avoided and large-scale antitoxin production is facilitated.[0351]

d) Anti-Recombinant Toxin A Neutralization of Toxin A Hemagglutination Activity In Vitro[0352]

Toxin A has hemagglutinating activity besides cytotoxic and enterotoxin properties. Specifically, toxin A agglutinates rabbit erythrocytes by binding to a trisaccharide (gal 1-3B1-4GIcNAc) on the cell surface. [H. Krivan et al., Infect. Immun., 53:573-581 (1986).] We examined whether the anti-recombinant toxin A (immune IgY, antibodies raised against the insoluble product expressed in pET) can neutralize the hemagglutination activity of toxin A in vitro. The hemagglutination assay procedure used was described by H. C. Krivan et al. Polyethylene glycol-fractionated immune or preimmune IgY were pre-absorbed with citrated rabbit erythrocytes prior to performing the hemagglutination assay because we have found that IgY alone can agglutinate red blood cells. Citrated rabbit red blood cells (RRBC's)(Cocalico) were washed twice by centrifugation at 450×g with isotonic buffer (0.1 M Tris-HCl, 0.05 M NaCl, pH 7.2). RRBC-reactive antibodies in the IgY were removed by preparing a 10% RRBC suspension (made by adding packed cells to immune or preimmune IgY) and incubating the mixture for 1 hour at 37° C. The RRBCs were then removed by centrifugation. Neutialization of the hemagglutination activity of toxin A by antibody was tested in round-bottomed 96-well microtiter plates. Twenty-five μl of toxin A (36 μg/ml) (Tech Lab) in isotonic buffer was mixed with an equal volume of different dilutions of immune or preimmune IgY in isotonic buffer, and incubated for 15 minutes at room temperature. Then, 50 μl of a 1% RRBC suspension in isotonic buffer was added and the mixture was incubated for 3 hours at 4° C. Positive control wells containing the final concentration of 9 μg/ml of toxin A after dilution without IgY were also included. Hemagglutination activity was assessed visually, with a diffuse matrix of RRBC's coating the bottom of the well representing a positive hemagglutination reaction and a tight button of RRBC's at the bottom of the well representing a negative reaction. The anti-recombinant immune IgY neutralized toxin A hemagglutination activity, giving a neutralization titer of 1:8. However, preimmune IgY was unable to neutralize the hemagglutination ability of toxin A.[0353]

e) Assay of In Vitro Toxin A Neutralizing Activity[0354]

The ability of the anti-recombinant toxin A IgY (immune IgY antibodies raised against pMA870-2680, the soluble recombinant binding domain protein expressed in pMAL, designated as Anti-tox. A-2 in FIG. 14, and referred to as recombinant region 6) and pre-immune IgY, prepared as described in Example 8(c) above, to neutralize the cytotoxic activity of toxin A was assessed in vitro using the CHO cell cytotoxicity assay, and toxin A (Tech Lab) at a concentration of 0.1 μg/ml, as described in Example 8(d) above. As additional controls, the anti-native toxin A IgY (CTA) and pre-immune IgY preparations described in Example 8(c) above were also tested. The results are shown in FIG. 14.[0355]

The anti-recombinant toxin A IgY demonstrated only partial neutralization of the cytotoxic activity of toxin A, while the pre-immune IgY did not demonstrate any significant neutralizing activity.[0356]

Example 14

In Vivo Neutralization of[0357]C. difficileToxin A

The ability of avian antibodies (IgY) raised against recombinant toxin A binding domain to neutralize the enterotoxin activity of[0358]C. difficiletoxin A was evaluated in vivo using Golden Syrian hamsters. The Example involved:

(a) preparation of the avian anti-recombinant toxin A IgY for oral administration;[0359]

(b) in vivo protection of hamsters from[0360]C. difficiletoxin A enterotoxicity by treatment of toxin A with avian anti-recombinant toxin A IgY; and (c) histologic evaluation of hamster ceca.

a) Preparation of the Avian Anti-Recombinant Toxin A IgY For Oral Administration[0361]

Eggs were collected from hens which had been immunized with the recombinant[0362]C. difficiletoxin A fragment pMA1870-2680 (described in Example 13, above). A second group of eggs purchased at a local supermarket was used as a preimmune (negative) control. Egg yolk immunoglobulin (IgY) was extracted by PEG from the two groups of eggs as described in Example 8(c), and the final IgY pellets were solubilized in one-fourth the original yolk volume using 0.1 M carbonate buffer (mixture of NaHCO, and Na₂CO₃), pH 9.5. The basic carbonate buffer was used in order to protect the toxin A from the acidic pH of the stomach environment.

b) In Vivo Protection of Hamsters Against[0363]C. difficileToxin A Enterotoxicity by Treatment of Toxin A with Avian Anti-recombinant Toxin A IgY

In order to assess the ability of the avian anti-recombinant toxin A IgY, prepared in section (a) above to neutralize the in vivo enterotoxin activity of toxin A, an in vivo toxin neutralization model was developed using Golden Syrian hamsters. This model was based on published values for the minimum amount of toxin A required to elicit diarrhea (0.08 mg toxin A/Kg body wt.) and death (0.16 mg toxin A/Kg body wt.) in hamsters when administered orally (Lyerly et al. Infect. Immun., 47:349-352 (1985).[0364]

For the study, four separate experimental groups were used, with each group consisting of 7 female Golden Syrian hamsters (Charles River), approx. three and one-half weeks old, weighing approx. 50 gms each. The animals were housed as groups of 3 and 4, and were offered food and water ad libitum through the entire length of the study.[0365]

For each animal, a mixture containing either 10 μg of toxin A (0.2 mg/Kg) or 30 μg of toxin A (0.6 mg/Kg) ([0366]C. difficiletoxin A was obtained from Tech Lab and 1 ml of either the anti-recombinant toxin A IgY or pre-immune IgY (from section (a) above) was prepared. These mixtures were incubated at 37° C. for 60 min. and were then administered to the animals by the oral route. The animals were then observed for the onset of diarrhea and death for a period of 24 hrs. following the administration of the toxin A+lgY mixtures, at the end of which time, the following results were tabulated and shown in Table 17:

TABLE 17


Study Outcome At 24 Hours

Study Outcome at 24 Hours

Experimental Group	Healthy¹	Diarrhea²	Dead³

10 μg Toxin A +Antitoxin Against	7	0	0
Interval 6
30 μg Toxin A +Antitoxin Against	7	0	0
Interval 6
10 μg Toxin A +Pre-Immune Serum	0	5	2
30 μg Toxin A +Pre-Immune	0	5	2

Pretreatment of toxin A at both doses tested, using the anti-recombinant toxin A IgY, prevented all overt symptoms of disease in hamsters. Therefore, pretreatment of[0367]C. difficiletoxin A, using the anti-recombinant toxin A IgY, neutralized the in vivo enterotoxin activity of the toxin A. In contrast, all animals from the two groups which received toxin A which had been pretreated using pre-immune IgY developed disease symptoms which ranged from diarrhea to death. The diarrhea which developed in the 5 animals which did not die in each of the two pre-imniune groups, spontaneously resolved by the end of the 24 hr. study period.

c) Histologic Evaluation of Hamster Ceca[0368]

In order to further assess the ability of anti-recombinant toxin A IgY to protect hamsters from the enterotoxin activity of toxin A, histologic evaluations were performed on the ceca of hamsters from the study described in section (b) above.[0369]

Three groups of animals were sacrificed in order to prepare histological specimens. The first group consisted of a single representative animal taken from each of the 4 groups of surviving hamsters at the conclusion of the study described in section (b) above. These animals represented the 24 hr. timepoint of the study.[0370]

The second group consisted of two animals which were not part of the study described above, but were separately treated with the same toxin A+pre-immune IgY mixtures as described for the animals in section (b) above. Both of these hamsters developed diarrhea, and were sacrificed 8 hrs. after the time of administration of the toxin A+pre-immune IgY mixtures. At the time of sacrifice, both animals were presenting symptoms of diarrhea. These animals represented the acute phase of the study.[0371]

The final group consisted of a single untreated hamster from the same shipment of animals as those used for the two previous groups. This animal served as the normal control.[0372]

Samples of cecal tissue were removed from the 7 animals described above, and were fixed overnight at 4° C. using 10% buffered formalin. The fixed tissues were paraffin-embedded, sectioned, and mounted on glass microscope slides. The tissue sections were then stained using hematoxylin and eosin (H and E stain), and were examined by light microscopy.[0373]

The tissues obtained from the two 24 hr. animals which received mixtures containing either 10 μg or 30 μg of toxin A and anti-recombinant toxin A IgY were indistinguishable from the normal control, both in terms of gross pathology, as well as at the microscopic level. These observations provide further evidence for the ability of anti-recombinant toxin A IgY to effectively neutralize the in vivo enterotoxin activity of[0374]C. difficiletoxin A, and thus its ability to prevent acute or lasting toxin A-induced pathology.

In contrast, the tissues from the two 24 hr. animals which received the toxin A+pre-immune IgY mixtures demonstrated significant pathology. In both of these groups, the mucosal layer was observed to be less organized than in the normal control tissue. The cytoplasm of the epithelial cells had a vacuolated appearance, and gaps were present between the epithelium and the underlying cell layers. The lamina propria was largely absent. Intestinal villi and crypts were significantly diminished, and appeared to have been overgrown by a planar layer of epithelial cells and fibroblasts. Therefore, although these animals overtly appeared to recover from the acute symptoms of toxin A intoxication, lasting pathologic alterations to the cecal mucosa had occurred.[0375]

The tissues obtained from the two acute animals which received mixtures of toxin A and pre-immune IgY demonstrated the most significant pathology. At the gross pathological level, both animals were observed to have severely distended ceca which were filled with watery, diarrhea-like material. At the microscopic level, the animal that was given the mixture containing 10 μg of toxin A and pre-immune IgY was found to have a mucosal layer which had a ragged, damaged appearance, and a disorganized, compacted quality. The crypts were largely absent, and numerous breaks in the epithelium had occurred. There was also an influx of erythrocytes into spaces between the epithelial layer and the underlying tissue. The animal which had received the mixture containing 30 μg of toxin A and pre-immune IgY demonstrated the most severe pathology. The cecal tissue of this animal had an appearance very similar to that observed in animals which had died from[0376]C. difficiledisease. Widespread destruction of the mucosa was noted, and the epithelial layer had sloughed. Hemorrhagic areas containing large numbers of erythrocytes were very prevalent. All semblance of normal tissue architecture was absent from this specimen. In terms of the presentation of pathologic events, this in vivo hamster model of toxin A-intoxication correlates very closely with the pathologic consequences ofC. dificiledisease in hamsters. The results presented in this Example demonstrate that while anti-recombinant toxin A (Interval 6) IgY is capable of only partially neutralizing the cytotoxic activity ofC. difficiletoxin A, the same antibody effectively neutralizes 100% of the in vivo enterotoxin activity of the toxin. While it is not intended that this invention be limited to this mechanism, this may be due to the cytotoxicity and enterotoxicity ofC. difficileToxin A as two separate and distinct biological functions.

Example 15In Vivo Neutralization ofC. difficileToxin A by Antibodies Against Recombinant Toxin A Polypeptides

The ability of avian antibodies directed against the recombinant[0377]C. difficiletoxin A fragment 1870-2680 (as expressed by pMA1870-2680; see Example 13) to neutralize the enterotoxic activity of toxin A was demonstrated in Example 14. The ability of avian antibodies (IgYs) directed against other recombinant toxin A epitopes to neutralize native toxin A in vivo was next evaluated. This example involved: (a) the preparation of IgYs against recombinant toxin A polypeptides; (b) in vivo protection of hamsters against toxin A by treatment with anti-recombinant toxin A IgYs and (c) quantification of specific antibody concentration in CTA andInterval 6 IgY PEG preparations.

The nucleotide sequence of the coding region of the entire toxin A protein is listed in SEQ ID NO:5. The amino acid sequence of the entire toxin A protein is listed in SEQ ID NO:6. The amino acid sequence consisting of amino acid residues 1870 through 2680 of toxin A is listed in SEQ ID NO:7. The amino acid sequence consisting of amino acid residues 1870 through 1960 of toxin A is listed in SEQ ID NO:8.[0378]

a) Preparation of IgY's Against Recombinant Toxin A Polypeptides[0379]

Eggs were collected from Leghorn hens which have been immunized with recombinant[0380]C. difficiletoxin A polypeptide fragments encompassing the entire toxin A protein. The polypeptide fragments used as immunogens were: 1) pMA 1870-2680 (Interval 6), 2) pPA 1100-1450 (Interval 4), and 3) a mixture of fragments consisting of pMA 30-300 (Interval 1), pMA 300-660 (Interval 2), pMA 660-1100 (Interval 3) and pMA 1450-1870 (Interval 5). This mixture of immunogens is referred to as Interval 1235. The location of each interval within the toxin A molecule is shown in FIG. 15A. In FIG. 15A, the following abbreviations are used: pP refers to the pET23 vector (New England BioLabs); pM refers to the pMAL™-c vector (New England BioLabs); A refers to toxin A; the numbers refer to the amino acid interval expressed in the clone. (For example, the designation pMA30-300 indicates that the recombinant clone encodes amino acids 30-300 of toxin A and the vector used was pMAL™-c).

The recombinant proteins were generated as described in Example 11. The IgYs were extracted and solubilized in 0.1M carbonate buffer pH 9.5 for oral administration as described in Example 14(a). The IgY reactivities against each individual recombinant interval was evaluated by ELISA as described in Example 13(c).[0381]

b) In Vivo Protection of Hamsters Against Toxin A by Treatment with Anti-Recombinant Toxin A Antibodies[0382]

The ability of antibodies raised against recombinant toxin A polypeptides to provide in vivo protection against the enterotoxic activity of toxin A was examined in the hamster model system. This assay was performed as described in Example 14(b). Briefly, for each 40-50 gram female Golden Syrian hamster (Charles River), 1 ml of[0383]IgY 4× (i.e., resuspended in ¼ of the original yolk volume) PEG prep againstInterval 6,Interval 4 or Interval 1235 was mixed with 30 μg (LD₁₀₀oral dose) ofC. difficiletoxin A (Tech Lab). Preimmune IgY mixed with toxin A served as a negative control. Antibodies raised againstC. difficiletoxoid A (Example 8) mixed with toxin A (CTA) served as a positive control. The mixture was incubated for 1 hour at 37° C. then orally administered to lightly etherized hamsters using an 18G feeding needle. The animals were then observed for the onset of diarrhea and death for a period of approximately 24 hours. The results are shown in Table 18.

TABLE 18


Study Outcome After 24 Hours

Treatment group	Healthy¹	Diarrhea²	Dead³

Preimmune	0	0	7
CTA	5	0	0
Interval 6	6	1	0
Interval 4	0	1	6
Interval 1235	0	0	7

Pre-treatment of toxin A with IgYs against[0384]Interval 6 prevented diarrhea in 6 of 7 hamsters and completely prevented death in all 7. In contrast, as with preimmune IgY, IgYs againstInterval 4 and Interval 1235 had no effect on the onset of diarrhea and death in the hamsters.

c) Quantification of Specific Antibody Concentration in CTA and[0385]Interval 6 IgY PEG Preparations

To determine the purity of IgY PEG preparations, an aliquot of a pMA1870-2680 (Interval 6) IgY PEG preparation was chromatographed using HPLC and a KW-803 sizing column (Shodex). The resulting profile of absorbance at 280 nm is shown in FIG. 16. The single large peak corresponds to the predicted MW of IgY. Integration of the area under the single large peak showed that greater than 95% of the protein eluted from the column was present in this single peak. This result demonstrated that the majority (>95%) of the material absorbing at 280 nm in the PEG preparation corresponds to IgY. Therefore, absorbance at 280 mm can be used to determine the total antibody concentration in PEG preparations.[0386]

To determine the concentration of Interval 6-specific antibodies (expressed as percent of total antibody) within the CTA and pMA1870-2680 (Interval 6) PEG preparations, defined quantities of these antibody preparations were affinity purified on a pPA1870-2680(H) (shown schematically in FIG. 15B) affinity column and the specific antibodies were quantified. In FIG. 15B the following abbreviations are used: pP refers to the pET23 vector (New England BioLabs); pM refers to the pMAL™-c vector (New England BioLabs); pG refers to the pGEX vector (Pharmacia); pB refers to the PinPoint™ Xa vector (Promega); A refers to toxin A; the numbers refer to the amino acid interval expressed in the clone. The solid black ovals represent the MBP; the hatched ovals represent glutathione S-transferase; the hatched circles represent the biotin tag; and HHH represents the poly-histidine tag.[0387]

An affinity column containing recombinant toxin A repeat protein was made as follows. Four ml of PBS-washed Actigel resin (Sterogene) was coupled with 5-10 mg of pPA1870-2680 inclusion body protein [prepared as described in Example (17) and dialyzed into PBS] in a 15 ml tube (Falcon) containing {fraction (1/10)} final volume Ald-coupling solution (1 M sodium cyanoborohydride). Aliquots of the supernatant from the coupling reactions, before and after coupling, were assessed by Coomassie staining of 7.5% SDS-PAGE gels. Based upon protein band intensities, greater than 6 mg of recombinant protein was coupled to the resin. The resin was poured into a 10 ml column (BioRad), washed extensively with PBS, pre-eluted with 4 M guanidine-HCl (in 10 mM Tris-HCl, pH 8.0; 0.005% thimerosal) and re-equilibrated with PBS. The column was stored at 4° C.[0388]

Aliquots of a pMA1870-2680 (Interval 6) or a CTA IgY polyclonal antibody preparation (PEG prep) were affinity purified on the above affinity column as follows. The column was attached to an UV monitor (ISCO) and washed with PBS. For pMA1870-2680 IgY purification, a 2×PEG prep (filter sterilized using a 0.45μ filter; approximately 500 mg total IgY) was applied. The column was washed with PBS until the baseline was re-established (the column flow-through was saved), washed with BBSTween to elute nonspecifically binding antibodies and re-equilibrated with PBS. Bound antibody was eluted from the column in 4 M guanidine-HCl (in 10 mM Tris-HCl, pH 8.0; 0.005% thimerosal). The entire elution peak was collected in a 15 ml tube (Falcon). The column was re-equilibrated and the column eluate was re-chromatographed as described above. The antibody preparation was quantified by UV absorbance (the elution buffer was used to zero the spectrophotometer). Total purified antibody was approximately 9 mg and 1 mg from the first and second chromatography passes, respectively. The low yield from the second pass indicated that most specific antibodies were removed by the first round of chromatography. The estimated percentage of[0389]Interval 6 specific antibodies in the pMA1870-2680 PEG prep is approximately 2%.

The percentage of[0390]Interval 6 specific antibodies in the CTA PEG prep was determined (utilizing the same column and methodology described above) to be approximately 0.5% of total IgY.

A 4× PEG prep contains approximately 20 mg/ml IgY. Thus in b) above, approximately 400 μg specific antibody in the[0391]Interval 6 PEG prep neutralized 30 μg toxin A in vivo.

Example 16In Vivo Treatment ofC. difficileDisease in Hamsters byRecombinant Interval 6 Antibodies

The ability of antibodies directed against[0392]recombinant Interval 6 of toxin A to protect hamsters in vivo fromC. difficiledisease was examined. This example involved: (a) prophylactic treatment ofC. difficiledisease and (b) therapeutic treatment ofC. difficiledisease.

a) Prophylactic Treatment of[0393]C. difficileDisease

This experiment was performed as described in Example 9(b). Three groups each consisting of 7 female 100 gram Syrian hamsters (Charles River) were prophylactically treated with either preimmune IgYs, IgYs against native toxin A and B [CTAB; see Example 8 (a) and (b)] or IgYs against[0394]Interval 6. IgYs were prepared as 4× PEG preparations as described in Example 9(a).

The animals were orally dosed 3 times daily, roughly at 4 hour intervals, for 12 days with 1 ml antibody preparations diluted in Ensure®. Using estimates of specific antibody concentration from Example 15(c), each dose of the[0395]Interval 6 antibody prep contained approximately 400 μg of specific antibody. Onday 2 each hamster was predisposed toC. difficileinfection by the oral administration of 3.0 mg of Clindamycin-HCl (Sigma) in 1 ml of water. Onday 3 the hamsters were orally challenged with 1 ml ofC. difficileinoculum strain ATCC 43596 in sterile saline containing approximately 100 organisms. The animals were then observed for the onset of diarrhea and subsequent death during the treatment period. The results are shown in Table 19.

TABLE 19


Lethality After 12 Days Of Treatment

Treatment Group	Number Animals Alive	NumberAnimals Dead

Preimmune
	0	7
CTAB	6	1
Interval 6	7	0

Treatment of hamsters with orally-administered IgYs against[0396]Interval 6 successfully protected 7 out of 7 (100%) of the animals fromC. difficiledisease. One of the hamsters in this group presented with diarrhea which subsequently resolved during the course of treatment. As shown previously in Example 9, antibodies to native toxin A and toxin B were highly protective. In this Example, 6 out of 7 animals survived in the CTAB treatment group. All of the hamsters treated with preimmune sera came down with diarrhea and died. The survivors in both the CTAB andInterval 6 groups remained healthy throughout a 12 day post-treatment period. In particular, 6 out of 7 Interval 6-treated hamsters survived at least 2 weeks after termination of treatment which suggests that these antibodies provide a long-lasting cure. These results represent the first demonstration that antibodies generated against a recombinant region of toxin A can prevent CDAD when administered passively to animals. These results also indicate that antibodies raised againstInterval 6 alone may be sufficient to protect animals fromC. difficiledisease when administered prophylactically.

Previously others had raised antibodies against toxin A by actively immunizing hamsters against a recombinant polypeptide located within the[0397]Interval 6 region [Lyerly, D. M., et al. (1990) Curr. Microbiol. 21:29]. FIG. 17 shows schematically the location of the Lyerly, et al. intra-Interval 6 recombinant protein (cloned into the pUC vector) in comparison with thecomplete Interval 6 construct (pMA1870-2680) used herein to generate neutralizing antibodies directed against toxin A. In FIG. 17, the solid black oval represents the MBP which is fised to thetoxin A Interval 6 in pMA1870-2680.

The Lyerly, et al antibodies (intra-Interval 6) were only able to partially protect hamsters against[0398]C. difficileinfection in terms of survival (4 out of 8 animals survived) and furthermore, these antibodies did not prevent diarrhea in any of the animals. Additionally, animals treated with the intra-Interval 6 antibodies [Lyerly, et al. (1990), supra] died when treatment was removed.

In contrast, the experiment shown above demonstrates that passive administration of anti-Interval 6 antibodies prevented diarrhea in 6 out of 7 animals and completely prevented death due to CDAD. Furthermore, as discussed above, passive administration of the anti-Interval 6 antibodies provides a long lasting cure (i.e., treatment could be withdrawn without incident).[0399]

b) Therapeutic Treatment of[0400]C. difficileDisease: In Vivo Treatment of an EstablishedC. diffcileInfection in Hamsters withRecombinant Interval 6 Antibodies

The ability of antibodies against[0401]recombinant interval 6 of toxin A to therapeutically treatC. difficiledisease was examined. The experiment was performed essentially as described in Example 10(b). Three groups, each containing seven to eight female Golden Syrian hamsters (100 g each; Charles River) were treated with either preimmune IgY, IgYs against native toxin A and toxin B (CTAB) and IgYs againstInterval 6. The antibodies were prepared as described above as 4× PEG preparations.

The hamsters were first predisposed to[0402]C. dificileinfection with a 3 mg dose of Clindamycin-HCl (Sigma) administered orally in 1 ml of water. Approximately 24 hrs later, the animals were orally challenged with 1 ml ofC. difficilestrain ATCC 43596 in sterile saline containing approximately 200 organisms. One day after infection, the presence of toxin A and B was determined in the feces of the hamsters using a commercial immunoassay kit (Cytoclone A+B EPA, Cambridge Biotech) to verify establishment of infection. Four members of each group were randomly selected and tested. Feces from an uninfected hamster was tested as a negative control. All infected animals tested positive for the presence of toxin according to the manufacturer's procedure. The initiation of treatment then started approximately 24 hr post-infection.

The animals were dosed daily at roughly 4 hr intervals with 1 ml antibody preparation diluted in Ensure® (Ross Labs). The amount of specific antibodies given per dose (determined by affinity purification) was estimated to be about 400 μg of anti-Interval 6 IgY (for animals in the[0403]Interval 6 group) and 100 μg and 70 μg of anti-toxin A (Interval 6-specific) and anti-toxin B (Interval 3-specific; see Example 19), respectively, for the CTAB preparation. The animals were treated for 9 days and then observed for an additional 4 days for the presence of diarrhea and death. The results indicating the number of survivors and the number of dead 4 days post-infection are shown in Table 20.

TABLE 20


In vivo Therapeutic Treatment WithInterval 6 Antibodies

Treatment Group	Number Animals Alive	NumberAnimals Dead

Preimmune
	4	3
CTAB	8	0
Interval 6	8	0

Antibodies directed against both[0404]Interval 6 and CTAB successfully prevented death fromC. difficilewhen therapeutically administered 24 hr after infection. This result is significant since many investigators begin therapeutic treatment of hamsters with existing drugs (e.g., vancomycin, phenelfamycins, tiacumicins, etc.) 8 hr post-infection [Swanson, et al. (1991) Antimicrobial Agents and Chemotherapy 35:1108 and (1989) J. Antibiotics 42:94].

Forty-two percent of hamsters treated with preimmune IgY died from CDAD. While the anti-Interval 6 antibodies prevented death in the treated hamsters, they did not eliminate all symptoms of CDAD as 3 animals presented with slight diarrhea. In addition, one CTAB-treated and one preimmune-treated animal also had[0405]diarrhea 14 days post-infection. These results indicate that anti-Interval 6 antibodies provide an effective means of therapy for CDAD.

Example 17Induction of Toxin A Neutralizing Antibodies RequiresSoluble Interval 6 Protein

As shown in Examples 11(d) and 15, expression of recombinant proteins in[0406]E. colimay result in the production of either soluble or insoluble protein. If insoluble protein is produced, the recombinant protein is solubilized prior to immunization of animals. To determine whether, one or both of the soluble or insoluble recombinant proteins could be used to generate neutralizing antibodies to toxin A, the following experiment was performed. This example involved a) expression of the toxin A repeats and subfragments of these repeats inE. coliusing a variety of expression vectors; b) identification of recombinant toxin A repeats and sub-regions to which neutralizing antibodies bind; and c) determination of the neutralization ability of antibodies raised against soluble and insoluble toxin A repeat immunogen.

a) Expression of the Toxin A Repeats and Subfragments of these Repeats in[0407]E. coliUsing a Variety of Expression Vectors

The[0408]Interval 6 immunogen utilized in Examples 15 and 16 was the pMA1870-2680 protein, in which the toxin A repeats are expressed as a soluble fusion protein with the MBP (described in Example 11). Interestingly, expression of this region (from the SpeI site to the end of the repeats, see FIG. 15B) in three other expression constructs, as either native (pPA1870-2680), poly-His tagged [pPA1870-2680 (H)] or biotin-tagged (pBA1870-2680) proteins resulted in completely insoluble protein upon induction of the bacterial host (see FIG. 15B). The host strain BL21 (Novagen) was used for expression of pBA1870-2680 and host strain BL21(DE3) (Novagen) was used for expression of pPA1870-2680 and pPA1870-2680(H). These insoluble proteins accumulated to high levels in inclusion bodies. Expression of recombinant plasmids inE. colihost cells grown in 2×YT medium was performed as described [Williams, et al. (1995), supra].

As summarized in FIG. 15B, expression of fragments of the toxin A repeats (as either N-terminal SpeI-EcoRI fragments, or C-terminal EcoRI-end fragments) also yielded high levels of insoluble protein using pGEX (pGA1870-2190), PinPoint™-Xa (pBA1870-2190 and pBA2250-2680) and pET expression systems (pPA1870-2190). The pGEX and pET expression systems are described in Example 11. The PinPoint™-Xa expression system drives the expression of fusion proteins in[0409]E. coli. Fusion proteins from PinPoint™-Xa vectors contain a biotin tag at the amino-terminal end and can be affinity purified SoftLink™ Soft Release avidin resin (Promega) under mild denaturing conditions (5 mM biotin).

The solubility of expressed proteins from the pPG1870-2190 and pPA1870-2190 expression constructs was determined after induction of recombinant protein expression under conditions reported to enhance protein solubility [These conditions comprise growth of the host at reduced temperature (30° C.) and the utilization of high (1 mM IPTG) or low (0.1 mM IPTG) concentrations of inducer [Williams et al. (1995), supra]. All expressed recombinant toxin A protein was insoluble under these conditions. Thus, expression of these fragments of the toxin A repeats in pET and pGEX expression vectors results in the production of insoluble recombinant protein even when the host cells are grown at reduced temperature and using lower concentrations of the inducer. Although expression of these fragments in pMal vectors yielded affinity purifiable soluble fusion protein, the protein was either predominantly insoluble (pMA1870-2190) or unstable (pMA2250-2650). Attempts to solubilize expressed protein from the pMA1870-2190 expression construct using reduced temperature or lower inducer concentration (as described above) did not improve fusion protein solubility.[0410]

Collectively, these results demonstrate that expression of the toxin A repeat region in[0411]E. coliresults in the production of insoluble recombinant protein, when expressed as either large (aa 1870-2680) or small (aa 1870-2190 or aa 2250-2680) fragments, in a variety of expression vectors (native or poly-his tagged pET, pGEX or PinPoint™-Xa vectors), utilizing growth conditions shown to enhance protein solubility. The exception to this rule were fusions with the MBP, which enhanced protein solubility, either partially (pMA1870-2190) or fully (pMA1870-2680).

b) Identification of Recombinant Toxin A Repeats and Sub-Regions to Which Neutralizing Antibodies Bind[0412]

Toxin A repeat regions to which neutralizing antibodies bind were identified by utilizing recombinant toxin A repeat region proteins expressed as soluble or insoluble proteins to deplete protective antibodies from a polyclonal pool of antibodies against native[0413]C. difficiletoxin A. An in vivo assay was developed to evaluate proteins for the ability to bind neutralizing antibodies.

The rational for this assay is as follows. Recombinant proteins were first pre-mixed with antibodies against native toxin A (CTA antibody; generated in Example 8) and allowed to react. Subsequently,[0414]C. difficiletoxin A was added at a concentration lethal to hamsters and the mixture was administered to hamsters via IP injection. If the recombinant protein contains neutralizing epitopes, the CTA antibodies would lose their ability to bind toxin A resulting in diarrhea and/or death of the hamsters.

The assay was performed as follows. The lethal dose of toxin A when delivered orally to nine 40 to 50 g Golden Syrian hamsters (Sasco) was determined to be 10 to 30 μg. The PEG-purified CTA antibody preparation was diluted to 0.5× concentration (i.e., the antibodies were diluted at twice the original yolk volume) in 0.1 M carbonate buffer, pH 9.5. The antibodies were diluted in carbonate buffer to protect them from acid degradation in the stomach. The concentration of 0.5× was used because it was found to be the lowest effective concentration against toxin A. The concentration of Interval 6-specific antibodies in the 0.5× CTA prep was estimated to be 10-15 μg/ml (estimated using the method described in Example 15).[0415]

The inclusion body preparation [[0416]insoluble Interval 6 protein; pPA 1870-2680(H)] and thesoluble Interval 6 protein [α]1870-2680; see FIG. 15] were both compared for their ability to bind to neutralizing antibodies againstC. difficiletoxin A (CTA). Specifically, 1 to 2 mg of recombinant protein was mixed with 5 ml of a 0.5×CTA antibody prep (estimated to contain 60-70 μg of Interval 6-specific antibody). After incubation for 1 hr at 37° C., CTA (Tech Lab) at a final concentration of 30 μg/ml was added and incubated for another 1 hr at 37° C. One ml of this mixture containing 30 μg of toxin A (and 10-15 μg of Interval 6-specific antibody) was administered orally to 40-50 g Golden Syrian hamsters (Sasco). Recombinant proteins that result in the loss of neutralizing capacity of the CTA antibody would indicate that those proteins contain neutralizing epitopes. Preimmune and CTA antibodies (both at 0.5×) without the addition of any recombinant protein served as negative and positive controls, respectively.

Two other inclusion body preparations, both expressed as insoluble products in the pET vector, were tested; one containing a different insert (toxin B fragment) other than[0417]Interval 6 called pPB1850-2070 (see FIG. 18) which serves as a control forinsoluble Interval 6, the other was a truncated version of theInterval 6 region called pPA1870-2190 (see FIG. 15B). The results of this experiment are shown in Table 21.

TABLE 21


Binding Of Neutralizing Antibodies BySoluble Interval
6 Protein Study Outcome After 24 Hours

Treatment Group¹	Healthy²	Diarrhea³	Dead⁴

Preimmune Ab	0	3	2
CTA Ab	4	1	0
CTA Ab + Int 6 (soluble)	1	2	2
CTA Ab + Int 6 (insoluble)	5	0	0
CTA Ab + pPB1850-2070	5	0	0
CTA Ab + pPA1870-2190	5	0	0

Preimmune antibody was ineffective against toxin A, while anti-CTA antibodies at a dilute 0.5× concentration almost completely protected the hamsters against the enterotoxic effects of CTA. The addition of recombinant proteins pPB1850-2070 or pPA1870-2190 to the anti-CTA antibody had no effect upon its protective ability, indicating that these recombinant proteins do not bind to neutralizing antibodies. On the other hand,[0418]recombinant Interval 6 protein was able to bind to neutralizing anti-CTA antibodies and neutralized the in vivo protective effect of the anti-CTA antibodies. Four out of five animals in the group treated with anti-CTA antibodies mixed withsoluble Interval 6 protein exhibited toxin associated toxicity (diarrhea and death). Moreover, the results showed thatInterval 6 protein must be expressed as a soluble product in order for it to bind to neutralizing anti-CTA antibodies since the addition ofinsoluble Interval 6 protein had no effect on the neutralizing capacity of the CTA antibody prep.

c) Determination of Neutralization Ability of Antibodies Raised Against Soluble and Insoluble Toxin A Repeat Immunogen[0419]

To determine if neutralizing antibodies are induced against solubilized inclusion bodies, insoluble toxin A repeat protein was solubilized and specific antibodies were raised in chickens. Insoluble pPA1870-2680 protein was solubilized using the method described in Williams et al. (1995), supra. Briefly, induced cultures (500 ml) were pelleted by centrifugation at 3,000×g for 10 min at 4° C. The cell pellets were resuspended thoroughly in 10 ml of inclusion body sonication buffer (25 mM HEPES pH 7.7, 100 mM KCl, 12.5 mM MgCl[0420]₂, 20% glycerol, 0.1% (v/v) Nonidet P-40, 1 mM DTT). The suspension was transferred to a 30 ml non-glass centrifuge tube. Five hundred μl of 10 mg/ml lysozyme was added and the tubes were incubated on ice for 30 min. The suspension was then frozen at −70° C. for at least 1 hr. The suspension was thawed rapidly in a water bath at room temperature and then placed on ice. The suspension was then sonicated using at least eight 15 sec bursts of the microprobe (Branson Sonicator Model No. 450) with intermittent cooling on ice.

The sonicated suspension was transferred to a 35 ml Oakridge tube and centrifuged at 6,000×g for 10 min at 4° C. to pellet the inclusion bodies. The pellet was washed 2 times by pipetting or vortexing in fresh, ice-cold RIPA buffer [0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate in TBS (25 mM Tris-Cl pH 7.5, 150 mM NaCl)]. The inclusion bodies were recentrifuged after each wash. The inclusion bodies were dried and transferred using a small metal spatula to a 15 ml tube (Falcon). One ml of 10% SDS was added and the pellet was solubilized by gently pipetting the solution up and down using a 1 ml micropipettor. The solubilization was facilitated by heating the sample to 95° C. when necessary.[0421]

Once the inclusion bodies were in solution, the samples were diluted with 9 volumes of PBS. The protein solutions were dialyzed overnight against a 100-fold volume of PBS containing 0.05% SDS at room temperature. The dialysis buffer was then changed to PBS containing 0.01% SDS and the samples were dialyzed for several hours to overnight at room temperature. The samples were stored at 4° C. until used. Prior to further use, the samples were warrned to room temperature to allow any precipitated SDS to go back into solution.[0422]

The inclusion body preparation was used to immunize hens. The protein was dialyzed into PBS and emulsified with approximately equal volumes of CFA for the initial immunization or IFA for subsequent booster immunizations. On day zero, for each of the recombinant recombinant preparations, two egg laying white Leghorn hens were each injected at multiple sites (IM and SC) with 1 ml of recombinant protein-adjuvant mixture containing approximately 0.5-1.5 mg of recombinant protein. Booster immunizations of 1.0 mg were given of[0423]days 14 andday 28. Eggs were collected on day 32 and the antibody isolated using PEG as described in Example 14(a). High titers of toxin A specific antibodies were present (as assayed by ELISA, using the method described in Example 13). Titers were determined for both antibodies against recombinant polypeptides pPA1870-2680 and pMA1870-2680 and were found to be comparable at >1:62,500.

Antibodies against soluble Interval 6 (pMA1870-2680) and insoluble Interval 6 [(inclusion body), pPA1870-2680] were tested for neutralizing ability against toxin A using the in vivo assay described in Example 15(b). Preimmune antibodies and antibodies against toxin A (CTA) served as negative and positive controls, respectively. The results are shown in Table 22.[0424]

TABLE 22


Neutralization Of Toxin A By Antibodies Against
Soluble Interval 6 Protein Study Outcome After 24 Hours

Antibody Treatment Group	Healthy¹	Diarrhea²	Dead³

Preimmune	1	0	4
CTA	5	0	0
Interval 6 (Soluble)⁴	5	0	0
Interval 6 (Insoluble)	0	2	3

Antibodies raised against native toxin A were protective while preimmune antibodies had little effect. As found using the in vitro CHO assay [described in Example 8(d)] where antibodies raised against the[0425]soluble Interval 6 could partially neutralize the effects of toxin A, here they were able to completely neutralize toxin A in vivo. In contrast, the antibodies raised against theinsoluble Interval 6 was unable to neutralize the effects of toxin A in vivo as shown above (Table 22) and in vitro as shown in the CHO assay [described in Example 8(d)].

These results demonstrate that soluble toxin A repeat immunogen is necessary to induce the production of neutralizing antibodies in chickens, and that the generation of such soluble immunogen is obtained only with a specific expression vector (pMal) containing the toxin A region spanking aa 1870-2680. That is to say, insoluble protein that is subsequently solubilized does not result in a toxin A antigen that will elicit a neutralizing antibody.[0426]

Example 18Cloning and Expression of theC. difficileToxin B Gene

The toxin B gene has been cloned and sequenced; the amino acid sequence deduced from the cloned nucleotide sequence predicts a MW of 269.7 kD for toxin B (Barroso et al., Nucl. Acids Res. 18:4004 (1990)]. The nucleotide sequence of the coding region of the entire toxin B gene is listed in SEQ ID NO:9. The amino acid sequence of the entire toxin B protein is listed in SEQ ID NO:10. The amino acid sequence consisting of[0427]amino acid residues 1850 through 2360 of toxin B is listed in SEQ ID NO:11. The amino acid sequence consisting ofamino acid residues 1750 through 2360 of toxin B is listed in SEQ ID NO:12.

Given the expense and difficulty of isolating native toxin B protein, it would be advantageous to use simple and inexpensive procaryotic expression systems to produce and purify high levels of recombinant toxin B protein for immunization purposes. Ideally, the isolated recombinant protein would be soluble in order to preserve native antigenicity, since solubilized inclusion body proteins often do not fold into native conformations. Indeed as shown in Example 17, neutralizing antibodies against recombinant toxin A were only obtained when soluble recombinant toxin A polypeptides were used as the immunogen. To allow ease of purification, the recombinant protein should be expressed to levels greater than 1 mg/liter of[0428]E. coliculture.

To determine whether high levels of recombinant toxin B protein could be produced in[0429]E. coli, fragments of the toxin B gene were cloned into various prokaryotic expression vectors, and assessed for the ability to express recombinant toxin B protein inE. coli. This Example involved (a) cloning of the toxin B gene and (b) expression of the toxin B gene inE. coli.

a) Cloning of the Toxin B Gene[0430]

The toxin B gene was cloned using PCR amplification from[0431]C. difficilegenomic DNA. Initially, the gene was cloned in two overlapping fragments, using primer pairs P5/P6 and P7/P8. The location of these primers along the toxin B gene is shown schematically in FIG. 18. The sequence of each of these primers is: P5: 5′TAGAAAAAATGGCAAATGT 3′ (SEQ ID NO:11); P6: 5′TTTCATCTTGTA GAGTCAAAG 3′ (SEQ ID NO:12); P7: 5′GATGCCACAAGATGATTTAGTG 3′ (SEQ ID NO:13); and P8: 5′CTAAT#GAGCTGTATCAGGATC 3′ (SEQ ID NO:14).

FIG. 18 also shows the location of the following primers along the toxin B gene: P9 which consists of the[0432]sequence 5′CGGAATTCCTAGAAAAAATGGCAA ATG 3′ (SEQ ID NO:15); P10 which consists of thesequence 5′GCTCTAGAATGA CCATAAGCTAGCCA 3′ (SEQ ID NO:16); P11 which consists of thesequence 5′CGGAATTCGAGTTGGTAGAAAGGTGGA 3′ (SEQ ID NO:17); P13 which consists of thesequence 5′CGGAATTCGGTTATTATCTTAAGGATG 3′ (SEQ ID NO:18); and P14 which consists of thesequence 5′CGGAATTCTTGATAACTGGAT TTGTGAC 3′ (SEQ ID NO:19). The amino acid sequence consisting of amino acid residues 1852 through 2362 of toxin B is listed in SEQ ID NO:20. The amino acid sequence consisting of amino acid residues 1755 through 2362 of toxin B is listed in SEQ ID NO:21.

[0433]Clostridium difficileVPI strain 10463 was obtained from the American Type Culture Collection (ATCC 43255) and grown under anaerobic conditions in brain-heart infusion medium (Becton Dickinson). High molecular-weightC. difficileDNA was isolated essentially as described [Wren and Tabaqchali (1987) J. Clin. Microbiol., 25:2402], except 1) 100 μg/ml proteinase K in 0.5% SDS was used to disrupt the bacteria and 2) cetytrimethylammonium bromide (CTAB) precipitation [as described by Ausubel et al., Eds.,Current Protocols in Molecular Biology, Vol. 2 (1989) Current Protocols] was used to remove carbohydrates from the cleared lysate. Briefly, after disruption of the bacteria with proteinase K and SDS, the solution is adjusted to approximately 0.7 M NaCl by the addition of a {fraction (1/7)} volume of 5M NaCl. A {fraction (1/10)} volume of CTAB/NaCl (10% CTAB in 0.7 M NaCl) solution was added and the solution was mixed thoroughly and incubated 10 min at 65° C. An equal volume of chloroform/isoamyl alcohol (24:1) was added and the phases were thoroughly mixed. The organic and aqueous phases were separated by centrifugation in a microfuge for 5 min. The aqueous supernatant was removed and extracted with phenol/chloroform/isoamyl alcohol (25:24:1). The phases were separated by centrifugation in a microfuge for 5 min. The supernatant was transferred to a fresh tube and the DNA was precipitated with isopropanol. The DNA precipitate was pelleted by brief centrifugation in a microfuge. The DNA pellet was washed with 70% ethanol to remove residual CTAB. The DNA pellet was then dried and redissolved in TE buffer (10 mM Tris-HCl pH8.0, 1 mM EDTA). The integrity and yield of genomic DNA was assessed by comparison with a serial dilution of uncut lambda DNA after electrophoresis on an agarose gel.

Toxin B fragments were cloned by PCR utilizing a proofreading thermostable DNA polymerase [native Pfu polymerase (Stratagene)]. The high fidelity of this polymerase reduces the mutation problems associated with amplification by error prone polymerases (e.g., Taq polymerase). PCR amplification was performed using the PCR primer pairs P5 (SEQ ID NO:11) with P6 (SEQ ID NO:12) and P7 (SEQ ID NO:13) with P8 (SEQ ID NO:14) in 50 μl reactions containing 10 mM Tris-HCl pH8.3, 50 mM KCl, 1.5 mM MgCl[0434]₂, 200 μM of each dNTP, 0.2 μM each primer, and 50 ngC. difficilegenomic DNA. Reactions were overlaid with 100 μl mineral oil, heated to 94° C. for 4 min, 0.51 μl native Pfu polymerase (Stratagene) was added, and the reactions were cycled 30 times at 94° C. for 1 min, 50° C. for 1 min, 72° C. (2 min for each kb of sequence to be amplified), followed by 10 min at 72° C. Duplicate reactions were pooled, chloroform extracted, and ethanol precipitated. After washing in 70% ethanol, the pellets were resuspended in 50 μl TE buffer (10 mM Tris-HCl pH8.0, 1 mM EDTA).

The P5/P6 amplification product was cloned into pUC19 as outlined below. 10 μl aliquots of DNA were gel purified using the Prep-a-Gene kit (BioRad), and ligated to SmaI restricted pUC19 vector. Recombinant clones were isolated and confirmed by restriction digestion using standard recombinant molecular biology techniques (Sambrook et al., 1989). Inserts from two independent isolates were identified in which the toxin B insert was oriented such that the vector BamHI and SacI sites were 5′ and 3′ oriented, respectively (pUCB10-1530). The insert-containing BamHI/SacI fragment was cloned into similarly cut pET23a-c vector DNA, and protein expression was induced in small scale cultures (5 ml) of identified clones.[0435]

Total protein extracts were isolated, resolved on SDS-PAGE gels, and toxin B protein identified by Western analysis utilizing a goat anti-toxin B affinity purified antibody (Tech Lab). Procedures for protein induction, SDS-PAGE, and Western blot analysis were performed as described in Williams et al. (1995), supra. In brief, 5 ml cultures of bacteria grown in 2XYT containing 100 μg/ml ampicillin containing the appropriate recombinant clone were induced to express recombinant protein by addition of IPTG to 1 mM. The[0436]E. colihosts used were: BL21(DE3) or BL21(DE3)LysS (Novagen) for pET plasmids.

Cultures were induced by the addition of IPTG to a final concentration of 1.0 mM when the cell density reached 0.5 OD[0437]₆₀₀, and induced protein was allowed to accumulate for two hrs after induction. Protein samples were prepared by pelleting 1 ml aliquots of bacteria by centrifugation (1 min in microfuge), and resuspension of the pelleted bacteria in 150 μl of 2× SDS-PAGE sample buffer (0.125 mM Tris-HCl pH 6.8, 2 mM EDTA, 6% SDS, 20% glycerol, 0.025% bromophenol blue; β-mercaptoethanol is added to 5% before use). The samples were heated to 95° C. for 5 min, then cooled and 5 or 10 μls loaded on 7.5% SDS-PAGE gels. High molecular weight protein markers (BioRad) were also loaded, to allow estimation of the MW of identified fusion proteins. After electrophoresis, protein was detected either generally by staining the gels with Coomassie Blue, or specifically, by blotting to nitrocellulose for Western blot detection of specific immunoreactive protein. The MW of induced toxin B reactive protein allowed the integrity of the toxin B reading frame to be determined.

The pET23b recombinant (pPB10-1530) expressed high MW recombinant toxin B reactive protein, consistent with predicted values. This confirmed that frame terminating errors had not occurred during PCR amplification. A pET23b expression clone containing the 10-1750aa interval of the toxin B gene was constructed, by fusion of the EcoRV-SpeI fragment of the P7/P8 amplification product to the P5-EcoRV interval of the P5/P6 amplification product (see FIG. 18) in pPB10-1530. The integrity of this clone (pPB10-1750) was confirmed by restriction mapping, and Western blot detection of expressed recombinant toxin B protein. Levels of induced protein from both pPB10-1530 and pPB10-1750 were too low to facilitate purification of usable amounts of recombinant toxin B protein. The remaining 1750-2360 aa interval was directly cloned into pMAL or pET expression vectors from the P7/P8 amplification product as described below.[0438]

b) Expression of the Toxin B Gene[0439]

i) Overview of Expression Methodologies[0440]

Fragments of the toxin B gene were expressed as either native or fusion proteins in[0441]E. coli. Native proteins were expressed in either the pET23a-c or pET16b expression vectors (Novagen). The pET23 vectors contain an extensive polylinker sequence in all three reading frames (a-c vectors) followed by a C-terminal poly-histidine repeat. The pET16b vector contains a N-terminal poly-histidine sequence immediately 5′ to a small polylinker. The poly-histidine sequence binds to Ni-Chelate columns and allows affinity purification of tagged target proteins [Williams et al. (1995), supra]. These affinity tags are small (10 aa for pET16b, 6 aa for pET23) allowing the expression and affinity purification of native proteins with only limited amounts of foreign sequences.

An N-terminal histidine-tagged derivative of pET16b containing an extensive cloning cassette was constructed to facilitate cloning of N-terminal poly-histidine tagged toxin B expressing constructs. This was accomplished by replacement of the promoter region of the pET23a and b vectors with that of the pET16b expression vector. Each vector was restricted with BglII and NdeI, and the reactions resolved on a 1.2% agarose gel. The pET16b promoter region (contained in a 200 bp BglII-NdeI fragment) and the promoter-less pET23 a or b vectors were cut from the gel, mixed and Prep-A-Gene (BioRad) purified. The eluted DNA was ligated, and transformants screened for promoter replacement by NcoI digestion of purified plasmid DNA (the pET16b promoter contains this site, the pET23 promoter does not). These clones (denoted pETHisa or b) contain the pET16b promoter (consisting of a pT7-lac promoter, ribosome binding site and poly-histidine (10aa) sequence) fused at the NdeI site to the extensive pET23 polylinker.[0442]

All MBP fusion proteins were constructed and expressed in the pMAL™-c or pMAL™-p2 vectors (New England Biolabs) in which the protein of interest is expressed as a C-terminal fusion with MBP. All pET plasmids were expressed in either the BL21(DE3) or BL21(DE3)LysS expression hosts, while pMal plasmids were expressed in the BL21 host.[0443]

Large scale (500 mls to 1 liter) cultures of each recombinant were grown in 2× YT broth, induced, and soluble protein fractions were isolated as described [Williams, et al. (1995), supra]. The soluble protein extracts were affinity chromatographed to isolate recombinant fusion protein, as described [Williams et al., (1995) supra]. In brief, extracts containing tagged pET fusions were chrornatographed on a nickel chelate column, and eluted using imidazole salts or low pH (pH 4.0) as described by the distributor (Novagen or Qiagen). Extracts containing soluble pMAL fusion protein were prepared and chromatographed in PBS buffer over an amylose resin (New England Biolabs) column, and eluted with PBS containing 10 mM maltose as described [Williams et al. (1995), supra].[0444]

ii) Overview of Toxin B Expression[0445]

In both large expression constructs described in (a) above, only low level (i.e., less than 1 mg/liter of intact or nondegraded recombinant protein) expression of recombinant protein was detected. A number of expression constructs containing smaller fragments of the toxin B gene were then constructed, to determine if small regions of the gene can be expressed to high levels (i.e., greater than 1 mg/liter intact protein) without extensive protein degradation. All were constructed by in frame fusions of convenient toxin B restriction fragments to either the pMAL-c, pET23a-c, pET 16b or pETHisa-b expression vectors, or by engineering restriction sites at specific locations using PCR amplification [using the same conditions described in (a) above]. In all cases, clones were verified by restriction mapping, and, where indicated, DNA sequencing.[0446]

Protein preparations from induced cultures of each of these constructs were analyzed, by SDS-PAGE, to estimate protein stability (Coomassie Blue staining) and immunoreactivity against anti-toxin B specific antiserum (Western analysis). Higher levels of intact (i.e., nondegraded), full length fusion proteins were observed with the smaller constructs as compared with the larger recombinants, and a series of expression constructs spanning the entire toxin B gene were constructed (FIGS. 18, 19 and[0447]20 and Table 23).

Constructs that expressed significant levels of recombinant toxin B protein (greater than 1 mg/liter intact recombinant protein) in[0448]E. coliwere identified and a series of these clones that spans the toxin B gene are shown in FIG. 19 and summarized in Table 23. These clones were utilized to isolate pure toxin B recombinant protein from the entire toxin B gene. Significant protein yields were obtained from pMAL expression constructs spanning the entire toxin B gene, and yields of full length soluble fusion protein ranged from an estimated 1 mg/liter culture (pMB 1100-1530) to greater than 20 mg/liter culture (pMB 1750-2360).

Representative purifications of MBP and poly-histidine-tagged toxin B recombinants are shown in FIGS. 21 and 22. FIG. 21 shows a Coomassie Blue stained 7.5% SDS-PAGE gel on which various protein samples extracted from bacteria harboring pMB1850-2360 were electrophoresed. Samples were loaded as follows: Lane 1: protein extracted from uninduced culture; Lane 2: induced culture protein; Lane 3: total protein from induced culture after sonication; Lane 4: soluble protein; and Lane 5: eluted affinity purified protein. FIG. 22 depicts the purification of recombinant proteins expressed in bacteria harboring either pPB1850-2360 (Lanes 1-3) or pPB1750-2360 (Lanes 4-6). Samples were loaded as follows: uninduced total protein ([0449]Lanes 1 and 4); induced total protein (Lanes 2 and 5); and eluted affinity purified protein (Lanes 3 and 6). The broad range molecular weight protein markers (BioRad) are shown inLane 7.

Thus, although high level expression was not attained using large expression constructs from the toxin B gene, usable levels of recombinant protein were obtained by isolating induced protein from a series of smaller pMAL expression constructs that span the entire toxin B gene.[0450]

These results represent the first demonstration of the feasibility of expressing recombinant toxin B protein to high levels in[0451]E. coli. As well, expression of small regions of the putative ligand binding domain (repeat region) of toxin B as native protein yielded insoluble protein, while large constructs, or fusions to MBP were soluble (FIG. 19), demonstrating that specific methodologies are necessary to produce soluble fusion protein from this interval.

iii) Clone Construction and Expression Details[0452]

A portion of the toxin B gene containing the toxin B repeat region [amino acid residues 1852-2362 of toxin B (SEQ ID NO:20)] was isolated by PCR amplification of this interval of the toxin B gene from[0453]C. difficilegenomic DNA. The sequence, and location within the toxin B gene, of the two PCR primers [P7 (SEQ ID NO:13) and P8 (SEQ ID NO:14)] used to amplify this region are shown in FIG. 18.

DNA from the PCR amplification was purified by chloroform extraction and ethanol precipitation as described above. The DNA was restricted with SpeI, and the cleaved DNA was resolved by agarose gel electrophoresis. The restriction digestion with SpeI cleaved the 3.6 kb amplification product into a 1.8 kb doublet band. This doublet band was cut from the gel and mixed with appropriately cut, gel purified pMALc or pET23b vector. These vectors were prepared by digestion with HindIII, filling in the overhanging ends using the Klenow enzyme, and cleaving with XbaI (pMALc) or NheI (pET23b). The gel purified DNA fragments were purified using the Prep-A-Gene kit (BioRad) and the DNA was ligated, transformed and putative recombinant clones analyzed by restriction mapping. pET and pMal clones containing the toxin B repeat insert (aa interval 1750-2360 of toxin B) were verified by restriction mapping, using enzymes that cleaved specific sites within the toxin B region. In both cases fusion of the toxin B SpeI site with either the compatible XbaI site (pMal) or compatible NheI site (pET) is predicted to create an in frame fusion. This was confirmed in the case of the pMB 1750-2360 clone by DNA sequencing of the clone junction and 5′ end of the toxin B insert using a MBP specific primer (New England Biolabs). In the case of the pET construct, the fusion of the blunt ended[0454]toxin B 3′ end to the filled HindIII site should create an in-frame fusion with the C-terminal poly-histidine sequence in this vector. The pPB1750-2360 clone selected had lost, as predicted, the HindIII site at this clone junction; this eliminated the possibility that an additional adenosine residue was added to the 3′ end of the PCR product by a terminal transferase activity of the Pfu polymerase, since fusion of this adenosine residue to the filled HindIII site would regenerate the restriction site (and was observed in several clones).

One liter cultures of each expression construct were grown, and fusion protein purified by affinity chromatography on either an amylose resin column (pMAL constructs; resin supplied by New England Biolabs) or Ni-chelate column (pET constructs; resin supplied by Qiagen or Novagen) as described [Williams et al. (1995), supra]. The integrity and purity of the fusion proteins were determined by Coomassie staining of SDS-PAGE protein gels as well as Western blot analysis with either an affinity purified goat polyclonal antiserum (Tech Lab), or a chicken polyclonal PEG prep, raised against the toxin B protein (CTB) as described above in Example 8. In both cases, affinity purification resulted in yields in excess of 20 mg protein per liter culture, of which greater than 90% was estimated to be full-length recombinant protein. It should be noted that the poly-histidine affinity tagged protein was released from the Qiagen Ni-NTA resin at low imidazole concentration (60 mM), necessitating the use of a 40 mM imidazole rather than a 60 mM imidazole wash step during purification.[0455]

A periplasmically secreted version of pMB1750-2360 was constructed by replacement of the promoter and MBP coding region of this construct with that from a related vector (pMAL™-p2; New England Biolabs) in which a signal sequence is present at the N-terminus of the MBP, such that fusion protein is exported. This was accomplished by substituting a BglII-EcoRV promoter fragment from pMAL-p2 into pMB1750-2360. The yields of secreted, affinity purified protein (recovered from osmotic shock extracts as described by Riggs in[0456]Current Protocols in Molecular Biology, Vol. 2, Ausubel, et al., Eds. (1989), Current Protocols, pp. 16.6.1-16.6.14] from this vector (pMBp1750-2360) were 6.5 mg/liter culture, of which 50% was estimated to be full-length fusion protein.

The interval was also expressed in two non-overlapping fragments. pMB1750-1970 was constructed by introduction of a frameshift into pMB 1750-2360, by restriction with HindIII, filling in the overhanging ends and religation of the plasmid. Recombinant clones were selected by loss of the HindIII site, and further restriction map analysis. Recombinant protein expression from this vector was more than 20 mg/liter of greater than 90% pure protein.[0457]

The complementary region was expressed in pMB1970-2360. This construct was created by removal of the 1750-1970 interval of pMB 1750-2360. This was accomplished by restriction of this plasmid with EcoRI (in the[0458]pMalc polylinker 5′ to the insert) and III, filling in the overhanging ends, and religation of the plasmid. The resultant plasmid, pMB1970-2360, was made using both intracellularly and secreted versions of the pMB1750-2360 vector.

No fusion protein was secreted in the pMBp1970-2360 version, perhaps due to a conformational constraint that prevents export of the fusion protein. However, the intracellularly expressed vector produced greater than 40 mg/liter of greater than 90% full-length fusion protein.[0459]

Constructs to precisely express the toxin B repeats in either pmalc (pMB1850-2360) or pET16b (pPB1850-2360) were constructed as follows. The DNA interval including the toxin B repeats was PCR amplified as described above utilizing PCR primers P14 (SEQ ID NO:19) and P8 (SEQ ID NO:14). Primer P14 adds a EcoRI site immediately flanking the start of the toxin B repeats.[0460]

The amplified fragment was cloned into the pT7 Blue T-vector (Novagen) and recombinant clones in which single copies of the PCR fragment were inserted in either orientation were selected (pT71850-2360) and confirmed by restriction mapping. The insert was excised from two appropriately oriented independently isolated pT71850-2360 plasmids, with EcoRI (5′ end of repeats) and PstI (in the flanking polylinker of the vector), and cloned into EcoRI/PstI cleaved pMalc vector. The resulting construct (pMB1850-2360) was confirmed by restriction analysis, and yielded 20 mg/l of soluble fusion protein [greater than 90% intact (i.e., nondegraded)] after affinity chromatography. Restriction of this plasmid with HindIII and religation of the vector resulted in the removal of the 1970-2360 interval. The resultant construct (pMB1850-1970) expressed greater than 70 mg/liter of 90% full length fusion protein.[0461]

The pPB1850-2360 construct was made by cloning a EcoRI (filled with Klenow)-BamHI fragment from a pT71850-2360 vector (opposite orientation to that used in the pMB1850-2360 construction) into NdeI (filled)/BamHI cleaved pET16b vector. Yields of affinity purified soluble fusion protein were 15 mg/liter, of greater than 90% full length fusion protein.[0462]

Several smaller expression constructs from the 1750-2070 interval were also constructed in His-tagged pET vectors, but expression of these plasmids in the BL21 (DE3) host resulted in the production of high levels of mostly insoluble protein (see Table 23 and FIG. 19). These constructs were made as follows.[0463]

pPB1850-1970 was constructed by cloning a BglII-HindIII fragment of pPB1850-2360 into BglII/HindIII cleaved pET23b vector. pPB1850-2070 was constructed by cloning a BglII-PvuII fragment of pPB1850-2360 into BglII/HincII cleaved pET23b vector. pPB1750-1970(c) was constructed by removal of the internal HindIII fragment of a pPB1750-2360 vector in which the vector HindIII site was regenerated during cloning (presumably by the addition of an A residue to the amplified PCR product by terminal transferase activity of Pfu polymerase). The pPB 1750-1970(n) construct was made by insertion of the insert containing the NdeI-HindIII fragment of pPB1750-2360 into identically cleaved pETHisb vector. All constructs were confirmed by restriction digestion.[0464]

An expression construct that directs expression of the 10-470 aa interval of toxin B was constructed in the pMalc vector as follows. A NheI (a[0465]site 5′ to the insert in the pET23 vector)-AflII (filled) fragment of the toxin B gene from pPB10-1530 was cloned into XbaI (compatible with NheI)/HindIII (filled) pMalc vector. The integrity of the construct (pMB10-470) was verified by restriction mapping and DNA sequencing of the 5′ clone junction using a MBP specific DNA primer (New England Biolabs). However, all expressed protein was degraded to the MBP monomer MW.

A second construct spanning this interval (aa 10-470) was constructed by cloning the PCR amplification product from a reaction containing the P9 (SEQ ID NO:15) and P10 (SEQ ID NO:16) primers (FIG. 18) into the pETHisa vector. This was accomplished by cloning the PCR product as an EcoRI-blunt fragment into EcoR] HincII restricted vector DNA; recombinant clones were verified by restriction mapping. Although this construct (pPB10-520) allowed expression and purification (utilizing the N-terminal polyhistidine affinity tag) of intact fusion protein, yields were estimated at less than 500 μg per liter culture.[0466]

Higher yield of recombinant protein from this interval (aa 10-520) were obtained by expression of the interval in two overlapping clones. The 10-330aa interval was cloned in both pETHisa and pMalc vectors, using the BamHI-AflIII (filled) DNA fragment from pPB10-520. This fragment was cloned into BamHI-HindIII (filled) restricted pMalc or BamHI-HincII restricted pETHisa vector. Recombinant clones were verified by restriction mapping. High level expression of either insoluble (pET) or soluble (pMal) fusion protein was obtained. Total yields of fusion protein from the pMB10-330 construct (FIG. 18) were 20 mg/liter culture, of which 10% was estimated to be full-length fusion protein. Although yields of this interval were higher and >90% full-length recombinant protein produced when expressed from the pET construct, the pMal fusion was utilized since the expressed protein was soluble and thus more likely to have the native conformation.[0467]

The pMB260-520 clone was constructed by cloning EcoRI-XbaI cleaved amplified DNA from a PCR reaction containing the P11 (SEQ ID NO:17) and P10 (SEQ ID NO:16) DNA primers (FIG. 18) into similarly restricted pMalc vector.[0468]

Yields of affinity purified protein were 10 mg/liter, of which approximately 50% was estimated to be full-length recombinant protein.[0469]

The aa510-1110 interval was expressed as described below. This entire interval was expressed as a pMal fusion by cloning the NheI-HindIII fragment of pUCB10-1530 into XbaI-HindIII cleaved pMalc vector. The integrity of the construct (pMB510-1110) was verified by restriction mapping and DNA sequencing of the 5′ clone junction using a MBP specific DNA primer. The yield of affinity purified protein was 25 mg/liter culture, of which 5% was estimated to be full-length fusion protein (1 mg/liter).[0470]

To attempt to obtain higher yields, this region was expressed in two fragments (aa510-820, and 820-1110) in the pMalc vector. The pMB510-820 clone was constructed by insertion of a SacI (in the[0471]pMalc polylinlcer 5′ to the insert)-HpaI DNA fragment from pMB510-1110 into SacI/StuI restricted pMalc vector. The pMB820-1110 vector was constructed by insertion of the HpaI-HindIII fragment of pUCB10-1530 into BamHI (filled)/HindIII cleaved pMalc vector. The integrity of these constructs were verified by restriction mapping and DNA sequencing of the 5′ clone junction using a MBP specific DNA primer.

Recombinant protein expressed from the pMB510-820 vector was highly unstable. However, high levels (20 mg/liter) of >90% full-length fusion protein were obtained from the pMB820-1105 construct. The combination of partially degraded pMB510-1110 protein (enriched for the 510-820 interval) with the pMB820-1110 protein provides usable amounts of recombinant antigen from this interval.[0472]

The aa1100-1750 interval was expressed as described below. The entire interval was expressed in the pMalc vector from a construct in which the AccI(filled)-SpeI fragment of pPB10-1750 was inserted into StuI/XbaI (XbaI is compatible with SpeI; StuI and filled AccI sites are both blunt ended) restricted pMalc. The integrity of this construct (pMB1100-1750) was verified by restriction mapping and DNA sequencing of the clone junction using a MBP specific DNA primer. Although 15 mg/liter of affinity purified protein was isolated from cells harboring this construct, the protein was greater than 99% degraded to MBP monomer size.[0473]

A smaller derivative of pMB1100-1750 was constructed by restriction of pMB1100-1750 with AflII and SalI (in the[0474]pMalc polylinker 3′ to the insert), filling in the overhanging ends, and religating the plasmid. The resultant clone (verified by restriction digestion and DNA sequencing) has deleted the aa1530-1750 interval, was designated pMB1100-1530. pMB1100-1530 expressed recombinant protein at a yield of greater than 40 mg/liter, of which 5% was estimated to be full-length fusion protein.

Three constructs were made to express the remaining interval. Initially, a BspHI (filled)-SpeI fragment from pPB10-1750 was cloned into EcoRI(filled)/XbaI cleaved pMalc vector. The integrity of this construct (pMB570-1750) was verified by restriction mapping and DNA sequencing of the 5′ clone junction using a MBP specific DNA primer. Expression of recombinant protein from this plasmid was very low, approximately 3 mg affinity purified protein per liter, and most was degraded to MBP monomer size. This region was subsequently expressed from a PCR amplified DNA fragment. A PCR reaction utilizing primers P13 [SEQ ID NO:18; P13 was engineered to introduce an[0475]EcoRI site 5′ to amplified toxin B sequences] and P8 (SEQ 10 ID NO:14) was performed onC. difficilegenomic DNA as described above. The amplified fragment was cleaved with EcoRI and SpeI, and cloned into EcoRI/XbaI cleaved pMalc vector. The resultant clone (pMB1530-1750) was verified by restriction map analysis, and recombinant protein was expressed and purified. The yield was greater than 20 mg protein per liter culture and it was estimated that 25% was full-length fusion protein; this was a significantly higher yield than the original pMB1570-1750 clone: The insert of pMB1530-1750 (in a EcoRI-SalI fragment) was transferred to the pETHisa vector (EcoRI/XhoI cleaved, XhoI and SalI ends are compatible). No detectable fusion protein was purified on Ni-Chelate columns from soluble lysates of cells induced to express fusion protein from this construct.

TABLE 23


Summary Of Toxin B Expression Constructs^a

Clone	Affinity Tag	Yield (mg/liter)	% Full Length

pPB10-1750	none	low (estimated from	?
		Western blot hyb)
pPB10-1530	none	low (as above)	?
pMB10-470	MBP	15 mg/l	0%
pPB10-520	poly-his	0.5 mg/l	20%
pPB10-330	poly-his	>20 mg/l (insoluble)	90%
pMB10-330	MBP	20 mg/l	10%
pMB260-520	MBP	10 mg/l	50%
pMB510-1110	MBP	25 mg/l	5%
pMB510-820	MBP	degraded (by Western
		blot hyb)
pMB820-1110	MBP	20 mg/l	90%
pMB1100-1750	MBP	15 mg/l	0%
pMB1100-1530	MBP	40 mg/l	5%
pMB1570-1750	MBP	3 mg/l	<5%
pPB1530-1750	poly-his	no purified protein	?
		detected
pMB1530-1750	MBP	20 mg/l	25%
pMB1750-2360	MBP	>20 mg/l	>90%
pMBp1750-2360	MBP	6.5 mg/l (secreted)	50%
pPB1750-2360	poly-his	>20 mg/l	>90%
pMB1750-1970	MBP	>20 mg/l	>90%
pMB1970-2360	MBP	40 mg/l	>90%
pMBp1970-2360	MBP	(no secretion)	NA
pMB1850-2360	MBP	20 mg/l	>90%
pPB1850-2360	poly-his	15 mg/l	>90%
pMB1850-1970	MBP	70 mg/l	>90%
pPB1850-1970	poly-his	>10 mg/l (insoluble)	>90%
pPB1850-2070	poly-his	>10 mg/l (insoluble)	>90%
pPB1750-1970(c)	poly-his	>10 mg/l (insoluble)	>90%
pPB1750-1970(n)	poly-his	>10 mg/l (insoluble)	>90%

Example 19Identification, Purification and Induction of Neutralizing Antibodies Against RecombinantC. difficileToxin B Protein

To determine whether recombinant toxin B polypeptide fragments can generate neutralizing antibodies, typically animals would first be immunized with recombinant proteins and anti-recombinant antibodies are generated. These anti-recombinant protein antibodies are then tested for neutralizing ability in vivo or in vitro. Depending on the immunogenic nature of the recombinant polypeptide, the generation of high-titer antibodies against that protein may take several months. To accelerate this process and identify which recombinant polypeptide(s) may be the best candidate to generate neutralizing antibodies, depletion studies were performed. Specifically, recombinant toxin B polypeptide were pre-screened by testing whether they have the ability to bind to protective antibodies from a CTB antibody preparation and hence deplete those antibodies of their neutralizing capacity. Those recombinant polypeptides found to bind CTB, were then utilized to generate neutralizing antibodies. This Example involved: a) identification of recombinant sub-regions within toxin B to which neutralizing antibodies bind; b) identification of toxin B sub-region specific antibodies that neutralize toxin B in vivo; and c) generation and evaluation of antibodies reactive to recombinant toxin B polypeptides.[0476]

a) Identification of Recombinant Sub-Regions within Toxin B to Which Neutralizing Antibodies Bind[0477]

Sub-regions within toxin B to which neutralizing antibodies bind were identified by utilizing recombinant toxin B proteins to deplete protective antibodies from a polyclonal pool of antibodies against native[0478]C. difficiletoxin B. An in vivo assay was developed to evaluate protein preparations for the ability to bind neutralizing antibodies. Recombinant proteins were first pre-mixed with antibodies directed against native toxin B (CTB antibody; see Example 8) and allowed to react for one hour at 37° C. Subsequently,C. difficiletoxin B (CTB; Tech Lab) was added at a concentration lethal to hamsters and incubated for another hour at 37° C. After incubation this mixture was injected intraperitoneally (IP) into hamsters. If the recombinant polypeptide contains neutralizing epitopes, the CTB antibodies will lose its ability to protect the hamsters against death from CTB. If partial or complete protection occurs with the CTB antibody-recombinant mixture, that recombinant contains only weak or non-neutralizing epitopes of toxin B. This assay was performed as follows.

Antibodies against CTB were generated in egg laying Leghorn hens as described in Example 8. The lethal dosage (LD[0479]₁₀₀) ofC. difficiletoxin B when delivered I.P. into 40 g female Golden Syrian hamsters (Charles River) was determined to be 2.5 to 5 μg. Antibodies generated against CTB and purified by PEG precipitation could completely protect the hamsters at the I.P. dosage determined above. The minimal amount of CTB antibody needed to afford good protection against 5 μg of CTB when injected I.P. into hamsters was also determined (1× PEG prep). These experiments defined the parameters needed to test whether a given recombinant protein could deplete protective CTB antibodies.

The cloned regions tested for neutralizing ability cover the entire toxin B gene and were designated as Intervals (INT) 1 through 5 (see FIG. 19). Approximately equivalent final concentrations of each recombinant polypeptide were tested. The following recombinant polypeptides were used: 1) a mixture of[0480]intervals 1 and 2 (INT-1,2); 2) a mixture ofIntervals 4 and 5 (INT-4, 5) and 3) Interval 3 (INT-3). Recombinant proteins (each at about 1 mg total protein) were first preincubated with a final CTB antibody concentration of 1× [i.e., pellet dissolved in original yolk volume as described in Example 1(c)] in a final volume of 5 mls for 1 hour at 37° C. Twenty-five μg of CTB (at a concentration of 5 μg/ml), enough CTB to kill 5 hamsters, was then added and the mixture was then incubated for 1 hour at 37° C. Five, 40 g female hamsters (Charles River) in each treatment group were then each given 1 ml of the mixture I.P. using a tuberculin syringe with a 27 gauge needle. The results of this experiment are shown in Table 24.

TABLE 24


Binding Of Neutralizing Antibodies ByINT 3 Protein

	Number Of	Number Of
Treatment Group¹	Animals Alive	AnimalsDead

CTB antibodies
3	2
CTB antibodies + INT1, 2	3	2
CTB antibodies + INT4, 5	3	2
CTB antibodies +INT 3	0	5

As shown in Table 24, the addition of recombinant proteins from INT-1, 2 or INT-4, 5 had no effect on the in vivo protective ability of the CTB antibody preparation compared to the CTB antibody preparation alone. In contrast, INT-3 recombinant polypeptide was able to remove all of the toxin B neutralizing ability of the CTB antibodies as demonstrated by the death of all the hamsters in that group.[0481]

The above experiment was repeated, using two smaller expressed fragments (pMB 1750-1970 and pMB 1970-2360, see FIG. 19) comprising the INT-3 domain to determine if that domain could be further subdivided into smaller neutralizing epitopes. In addition, full-length INT-3 polypeptide expressed as a nickel tagged protein (pPB1750-2360) was tested for neutralizing ability and compared to the original INT-3 expressed MBP fusion (pMB1750-2360). The results are shown in Table 25.[0482]

TABLE 25


Removal Of Neutralizing Antibodies By Repeat
Containing Proteins

	Number Of	Number Of
Treatment Group¹	Animals Alive	AnimalsDead

CTB antibodies
	5	0
CTB antibodies + pPB1750-2360	0	5
CTB antibodies + pMB1750-2360	0	5
CTB antibodies + pMB1970-2360	3	2
CTB antibodies + pMB1750-1970	2	3

The results summarized in Table 25 indicate that the smaller polypeptide fragments within the INT-3 domain, pMB1750-1970 and pMB1970-2360, partially lose the ability to bind to and remove neutralizing antibodies from the CTB antibody pool. These results demonstrate that the full length INT-3 polypeptide is required to completely deplete the CTB antibody pool of neutralizing antibodies. This experiment also shows that the neutralization epitope of rNT-3 can be expressed in alternative vector systems and the results are independent of the vector utilized or the accompanying fusion partner.[0483]

[0484]Other Interval 3 specific proteins were subsequently tested for the ability to remove neutralizing antibodies within the CTB antibody pool as described above. TheInterval 3 specific proteins used in these studies are summarized in FIG. 23. In FIG. 23 the following abbreviations are used: pP refers to the pET23 vector; pM refers to the pMALc vector; B refers to toxin B; the numbers refer to the amino acid interval expressed in the clone. The solid black ovals represent the MBP; and HHH represents the poly-histidine tag.

Only recombinant proteins comprising the entire toxin B repeat domain (pMB1750-2360, pPB1750-2360 and pPB1850-2360) can bind and completely remove neutralizing antibodies from the CTB antibody pool. Recombinant proteins comprising only a portion of the toxin B repeat domain were not capable of completely removing neutralizing antibodies from the CTB antibody pool (pMB1750-1970 and pMB1970-2360 could partially remove neutralizing antibodies while pMB1850-1970 and pPB1850-2070 failed to remove any neutralizing antibodies from the CTB antibody pool).[0485]

The above results demonstrate that only the complete ligand binding domain (repeat region) of the toxin B gene can birid and completely remove neutralizing antibodies from the CTB antibody pool. These results demonstrate that antibodies directed against the entire toxin B repeat region are necessary for in vivo toxin neutralization (see FIG. 23; only the recombinant proteins expressed by the pMB1750-2360, pPB1750-2360 and pPB1850-2360 vectors are capable of completely removing the neutralizing antibodies from the CTB antibody pool).[0486]

These results represent the first indication that the entire repeat region of toxin B would be necessary for the generation of antibodies capable of neutralizing toxin B, and that sub-regions may not be sufficient to generate maximal titers of neutralizing antibodies.[0487]

b) Identification of Toxin B Sub-Region Specific Antibodies that Neutralize Toxin B In Vivo[0488]

To determine if antibodies directed against the toxin B repeat region are sufficient for neutralization, region specific antibodies within the CTB antibody preparation were affinity purified, and tested for in vivo neutralization. Affinity columns containing recombinant toxin B repeat proteins were made as described below. A separate affinity column was prepared using each of the following recombinant toxin B repeat proteins: pPB1750-2360, pPB1850-2360, pMB1750-1970 and pMB1970-2360.[0489]

For each affinity column to be made, four ml of PBS-washed Actigel resin (Sterogene) was coupled overnight at room temperature with 5-10 mg of affinity purified recombinant protein (first extensively dialyzed into PBS) in 15 ml tubes (Falcon) containing a {fraction (1/10)} final volume Ald-coupling solution (1 M sodium cyanoborohydride). Aliquots of the supernatants from the coupling reactions, before and after coupling, were assessed by Coomassie staining of 7.5% SDS-PAGE gels. Based on protein band intensities, in all cases greater than 30% coupling efficiencies were estimated. The resins were poured into 10 ml columns (BioRad), washed extensively with PBS, pre-eluted with 4M guanidine-HCl (in 10 mM Tris-HCl, pH 8.0) and reequilibrated in PBS. The columns were stored at 4° C.[0490]

Aliquots of a CTB IgY polyclonal antibody preparation (PEG prep) were affinity purified on each of the four columns as described below. The columns were hooked to a UV monitor (ISCO), washed with PBS and 40 ml aliquots of a 2× PEG prep (filter sterilized using a 0.45 p filter) were applied. The columns were washed with PBS until the baseline value was re-established. The columns were then washed with BBStween to elute nonspecifically binding antibodies, and reequilibrated with PBS. Bound antibody was eluted from the column in 4M guanidine-HCl (in 10 nM Tris-HCl, pH8.0). The eluted antibody was immediately dialyzed against a 100-fold excess of PBS at 4° C. for 2 hrs. The samples were then dialyzed extensively against at least 2 changes of PBS, and affinity purified antibody was collected and stored at 4° C. The antibody preparations were quantified by UV absorbance. The elution volumes were in the range of 4-8 ml. All affinity purified stocks contained similar total antibody concentrations, ranging from 0.25-0.35% of the total protein applied to the columns.[0491]

The ability of the affinity purified antibody preparations to neutralize toxin B in vivo was determined using the assay outlined in a) above. Affinity purified antibody was diluted 1:1 in PBS before testing. The results are shown in Table 26.[0492]

TABLE 26


Neutralization Of Toxin B By Affinity Purified Antibodies

	Number	Number
	Animals	Animals
Treatment group^a	Alive^b	Dead^b

Preimmune¹	0	5
CTB¹; 400μg	5	0
CTB (AP on pPB1750-2360);²875μg	5	0
CTB (AP on pMB1750-1970);²875μg	5	0
CTB (AP on pMB1970-2360);²500μg	5	0


# received toxin premixed with antibody raised against the indicated protein, as either:¹4X antibody PEG prep or²affinity purified (AP) antibody (from CTB PEG prep, on indicated columns). The amount of specific antibody in each prep is indicated; the amount is directly determined for affinity purified preps and is estimated for the 4X
# CTB as described in Example 15.

The observation that antibodies affinity purified against the non-overlapping pMB1750-1970 and pMB1970-2360 proteins neutralized toxin B raised the possibility that either 1) antibodies specific to repeat sub-regions are sufficient to neutralize toxin B or 2) sub-region specific proteins can bind most or all repeat specific antibodies present in the CTB polyclonal pool. This would likely be due to conformational similarities between repeats, since homology in the primary amino acid sequences between different repeats is in the range of only 25-75% [Eichel-Streiber, et al. (1992) Molec. Gen. Genetics 233:260]. These possibilities were tested by affinity chromatography.[0494]

The CTB PEG prep was sequentially depleted 2× on the pMB1750-1970 column; only a small elution peak was observed after the second chromatography, indicating that most reactive antibodies were removed. This interval depleted CTB preparation was then chromatographed on the pPB1850-2360 column; no antibody bound to the column. The reactivity of the CTB and CTB (pMB1750-1970 depleted) preps to pPB1750-2360, pPB1850-2360, pMB1750-1970 and pMB1970-2360 proteins was then determined by ELISA using the protocol described in Example 13(c). Briefly, 96-well microtiter plates (Falcon, Pro-Bind Assay Plates) were coated with recombinant protein by adding 100 μl volumes of protein at 1-2 μg/ml in PBS containing 0.005% thimerosal to each well and incubating overnight at 4° C. The next morning, the coating suspensions were decanted and the wells were washed three times using PBS. In order to block non-specific binding sites, 100 μl of 1.0% BSA (Sigma) in PBS (blocking solution) was then added to each well, and the plates were incubated for 1 hr. at 37° C. The blocking solution was decanted and duplicate samples of 150 μl of diluted antibody was added to the first well of a dilution series. The initial testing serum dilution was (1/200 for CTB prep, (the concentration of depleted CTB was standardized by OD[0495]₂₈₀) in blocking solution containing 0.5% Tween 20, followed by 5-fold serial dilutions into this solution. This was accomplished by serially transferring 30 μl aliquots to 120 μl buffer, mixing, and repeating the dilution into a fresh well. After the final dilution, 30 μl was removed from the well such that all wells contained 120 μl final volume. A total of 5 such dilutions were performed (4 wells total). The plates were incubated for 1 hr at 37° C. Following this incubation, the serially diluted samples were decanted and the wells were washed three times using PBS containing 0.5% Tween 20 (PBST), followed by two 5 min washes using BBS-Tween and a final three washes using PBST. To each well, 100 μl of 1/1000 diluted secondary antibody [rabbit anti-chicken IgG alkaline phosphatase (Sigrna) diluted in blocking solution containing 0.5% Tween 20] was added, and the plate was incubated 1 hr at 37° C. The conjugate solutions were decanted and the plates were washed 6 times in PBST, then once in 50 mM Na₂CO₃, 10 nM MgCl₂, pH 9.5. The plates were developed by the addition of 100 μl of a solution containing 1 mg/ml para-nitro phenyl phosphate (Sigma) dissolved in 50 mM Na₂CO₃, 10 mM MgCl₂, pH9.5 to each well. The plates were then incubated at room temperature in the dark for 5-45 min. The absorbency of each well was measured at 410 nm using a Dynatech MR 700 plate reader.

As predicted by the affinity chromatography results, depletion of the CTB prep on the pMB1750-1970 column removed all detectable reactivity to the pMB1970-2360 protein. The reciprocal purification of a CTB prep that was depleted on the pMB1970-2360 column yielded no bound antibody when chromatographed on the pMB1750-1970 column. These results demonstrate that all repeat reactive antibodies in the CTB polyclonal pool recognize a conserved structure that is present in non-overlapping repeats. Although it is possible that this conserved structure represents rare conserved linear epitopes, it appears more likely that the neutralizing antibodies recognize a specific protein conformation. This conclusion was also suggested by the results of Western blot hybridization analysis of CTB reactivity to these recombinant proteins.[0496]

Western blots of 7.5% SDS-PAGE gels, loaded and electrophoresed with defined quantities of each recombinant protein, were probed with the CTB polyclonal antibody preparation. The blots were prepared and developed with alkaline phosphatase as described in Example 3. The results are shown in FIG. 24.[0497]

FIG. 24 depicts a comparison of inimunoreactivity of IgY antibody raised against either native or recombinant toxin B antigen. Equal amounts of pMB1750-1970 (lane 1), pMB1970-2360 (lane 2), pPB1850-2360 (lane 3) as well as a serial dilution of pPB1750-2360 (lanes 4-6 comprising 1×, 1/10× and 1/100× amounts, respectively) proteins were loaded in duplicate and resolved on a 7.5% SDS-PAGE gel. The gel was blotted and each half was hybridized with PEG prep IgY antibodies from chickens immunized with either native CTB or pPB1750-2360 protein. Note that the full-length pMB1750-1970 protein was identified only by antibodies reactive to the recombinant protein (arrows).[0498]

Although the CTB prep reacts with the pPB1750-2360, pPB1850-2360, and pMB1970-2360 proteins, no reactivity to the pMB1750-1970 protein was observed (FIG. 24). Given that all repeat reactive antibodies can be bound by this protein during affinity chromatography, this result indicates that the protein cannot fold properly on Western blots. Since this eliminates all antibody reactivity, it is unlikely that the repeat reactive antibodies in the CTB prep recognize linear epitopes. This may indicate that in order to induce protective antibodies, recombinant toxin B protein will need to be properly folded.[0499]

c) Generation and Evaluation of Antibodies Reactive to Recombinant Toxin B Polypeptides[0500]

i) Generation of Antibodies Reactive to Recombinant Toxin B Proteins[0501]

Antibodies against recombinant proteins were generated in egg laying Leghorn hens as described in Example 13. Antibodies were raised [using Freunds adjuvant (Gibco) unless otherwise indicated] against the following recombinant proteins: 1) a mixture of[0502]Interval 1+2 proteins (see FIG. 18); 2) a mixture of

interval

4 and 5 proteins (see FIG. 18); 3) pMB1970-2360 protein; 4) pPB1750-2360 protein; 5) pMB1750-2360; 6) pMB1750-2360 [Titermax adjuvant (Vaxcell)]; 7) pMB1750-2360 (Gerbu adjuvant (Biotech)]; 8) pMBp1750-2360 protein; 9) pPB1850-2360; and 10) pMB1850-2360.

Chickens were boosted at least 3 times with recombinant protein until ELISA reactivity [using the protocol described in b) above with the exception that the plates were coated with pPB1750-2360 protein] of polyclonal PEG preps was at least equal to that of the CTB polyclonal antibody PEG prep. ELISA titers were determined for the PEG preps from all of the above immunogens and were found to be comparable ranging from 1:12500 to 1:62500. High titers were achieved in all cases except in 6) pMB 1750-2360 in which strong titers were not observed using the Titermax adjuvant, and this preparation was not tested further.[0503]

ii) Evaluation of Antibodies Reactive to Recombinant Proteins by Western Blot Hybridization[0504]

Western blots of 7.5% SDS-PAGE gels, loaded and electrophoresed with defined quantities of recombinant protein (pMB1750-1970, pPB1850-2360, and pMB1970-2360 proteins and a serial dilution of the pPB1750-2360 to allow quantification of reactivity), were probed with the CTB, pPB1750-2360, pMB1750-2360 and pMB1970-2360 polyclonal antibody preparations (from chickens immunized using Freunds adjuvant). The blots were prepared and developed with alkaline phosphatase as described above in b).[0505]

As shown in FIG. 24, the CTB and pMB1970-2360 preps reacted strongly with the pPB1750-2360, pPB1850-2360, and pMB1970-2360 proteins while the pPB1750-2360 and pMB1970-2360 (Gerbu) preparations reacted strongly with all four proteins. The Western blot reactivity of the pPB1750-2360 and pMB1970-2360 (Gerbu) preparations were equivalent to that of the CTB preparation, while reactivity of the pMB1970-2360 preparation was <10% that of the CTB prep. Despite equivalent ELISA reactivities only weak reactivity (approximately 1%) to the recombinant proteins were observed in PEG preps from two independent groups immunized with the pMB1750-2360 protein and one group immunized with the pMB1750-2360 preparation using Freunds adjuvant.[0506]

Affinity purification was utilized to determine if this difference in immunoreactivity by Western blot analysis reflects differing antibody titers. Fifty[0507]ml 2× PEG preparations from chickens immunized with either pMB1750-2360 or pMB1970-2360 protein were chromatographed on the pPB1750-2360 affinity column from b) above, as described. The yield of affinity purified antibody (% total protein in preparation) was equivalent to the yield obtained from a CTB PEG preparation in b) above. Thus, differences in Western reactivity reflect a qualitative difference in the antibody pools, rather than quantitative differences. These results demonstrate that certain recombinant proteins are more effective at generating high affinity antibodies (as assayed by Western blot hybridization).

iii) In Vivo Neutralization of Toxin B Using Antibodies Reactive to Recombinant Protein[0508]

The in vivo hamster model [described in Examples 9 and 14(b)] was utilized to assess the neutralizing ability of antibodies raised against recombinant toxin B proteins. The results from three experiments are shown below in Tables 27-29.[0509]

The ability of each immunogen to neutralize toxin B in vivo has been compiled and is shown in Table 30. As predicted from the recombinant protein-CTB premix studies (Table 24) only antibodies to Interval 3 (1750-2366) and not the other regions of toxin B (i.e., intervals 1-5) are protective. Unexpectedly, antibodies generated to INT-3 region expressed in pMAL vector (pMB1750-2360 and pMpB1750-2360) using Freunds adjuvant were non-neutralizing. This observation is reproducible, since no neutralization was observed in two independent immunizations with pMB1750-2360 and one immunization with pMpB1750-2360. The fact that 5× quantities of affinity purified toxin B repeat specific antibodies from pMB1750-2360 PEG preps cannot neutralize toxin B while 1× quantities of affinity purified anti-CTB antibodies can (Table 28) demonstrates that the differential ability of CTB antibodies to neutralize toxin B is due to qualitative rather than quantitative differences in these antibody preparations. Only when this region was expressed in an alternative vector (pPB1750-2360) or using an alternative adjuvant with the pMB1750-2360 protein were neutralizing antibodies generated. Importantly, antibodies raised using Freunds adjuvant to pPB1850-2360, which contains a fragment that is only 100 amino acids smaller than recombinant pPB1750-2360, are unable to neutralize toxin B in vivo (Table 27); note also that the same vector is used for both pPB1850-2360 and pPB1750-2360.[0510]

TABLE 27


In Vivo Neutralization Of Toxin B

Treatment Group^a	Number Animals Alive^b	Number Animals Dead^b

Preimmune	0	5
CTB	5	0
INT1 + 2	0	5
INT 4 + 5	0	5
pMB1750-2360	0	5
pMB1970-2360	0	5
pPB1750-2360	5	0

[0511]

TABLE 28


In Vivo Neutralization Of Toxin B Using Affinity
Purified Antibodies

	Number Animals	Number Animals
Treatment Group^a	Alive^b	Dead^b

Preimmune(1)	0	5
CTB(1)	5	0
pPB1750-2360(1)	5	0
1.5 mg anti-pMB1750-2360(2)	1	4
1.5 mg anti-pMB1970-2360(2)	0	5
300 μg anti-CTB(2)	5	0


# either (1) 4X antibody PEG prep or (2) affinity purified antibody (on a pPB1750-2360 resin), either 1.5 mg/group (anti-pMB1750-2360 and anti-pMB1970-2360; used undiluted affinity purified antibody) or 350 μg/group (anti-CTB, repeat specific; used 1/5 diluted anti-CTB antibody).

[0512]

TABLE 29


Generation Of Neutralizing Antibodies Utilizing
The Gerbu Adjuvant

	Number Animals	Number Animals
Treatment Group^a	Alive^b	Dead^b

Preimmune	0	5
CTB	5	0
pMB1970-2360	0	5
pMB1850-2360	0	5
pPB1850-2360	0	5
pMB1750-2360 (Gerbu adj)	5	0

[0513]

TABLE 30


In Vivo Neutralization Of Toxin B

			Antigen	In vivo
		Tested	Utilized	Neutral-
Immunogen	Adjuvant	Preparation^a	For AP	ization^b

Preimmune	NA¹	PEG	NA	no
CTB (native)	Titermax	PEG	NA	yes
CTB (native)	Titermax	AP	pPB1750-2360	yes
CTB (native)	Titermax	AP	pPB1850-2360	yes
CTB (native)	Titermax	AP	pPB1750-1970	yes
CTB (native)	Titermax	AP	pPB1970-2360	yes
pMB1750-2360	Freunds	PEG	NA	no
pMB1750-2360	Freunds	AP	pPB1750-2360	no
pMB1750-2360	Gerbu	PEG	NA	yes
pMB1970-2360	Freunds	PEG	NA	no
pMB1970-2360	Freunds	AP	pPB1750-2360	no
pPB1750-2360	Freunds	PEG	NA	yes
pPB1850-2360	Freunds	PEG	NA	no
pMB1850-2360	Freunds	PEG	NA	no
INT 1 + 2	Freunds	PEG	NA	no
INT 4 + 5	Freunds	PEG	NA	no

The pPB1750-2360 antibody pool confers significant in vivo protection, equivalent to that obtained with the affinity purified CTB antibodies. This correlates with the observed high affinity of this antibody pool (relative to the pMB1750-2360 or pMB1970-2360 pools) as assayed by Western blot analysis (FIG. 24). These results provide the first demonstration that in vivo neutralizing antibodies can be induced using recombinant toxin B protein as immunogen.[0514]

The failure of high concentrations of antibodies raised against the pMB1750-2360 protein (using Freunds adjuvant) to neutralize, while the use of Gerbu adjuvant and pMB1750-2360 protein generates a neutralizing response, demonstrates that conformation or presentation of this protein is essential for the induction of neutralizing antibodies. These results are consistent with the observation that the neutralizing antibodies produced when native CTB is used as an immunogen appear to recognize conformational epitopes [see section b) above]. This is the first demonstration that the conformation or presentation of recombinant toxin B protein is essential to generate high titers of neutralizing antibodies.[0515]

Example 20Determination of Quantitative and Qualitative Differences Between pMB 1750-2360, pMB 750-2360 (Gerbu) or pPB1750-2360 IgY Polyclonal Antibody Preparations

In Example 19, it was demonstrated that toxin B neutralizing antibodies could be generated using specific recombinant toxin B proteins (pPB1750-2360) or specific adjuvants. Antibodies raised against pMB1750-2360 were capable of neutralizing the enterotoxin effect of toxin B when the recombinant protein was used to immunize hens in conjunction with the Gerbu adjuvant, but not when Freunds adjuvant was used. To determine the basis for these antigen and adjuvant restrictions, toxin B-specific antibodies present in the neutralizing and non-neutralizing PEG preparations were isolated by affinity chromatography and tested for qualitative or quantitative differences. The example involved a) purification of anti-toxin B specific antibodies from pMB1750-2360 and pPB1750-2360 PEG preparations and b) in vivo neutralization of toxin B using the affinity purified antibody.[0516]

a) Purification of specific Antibodies from pMB1750-2360 and pPB1750-2360 PEG Preparations[0517]

To purify and determine the concentration of specific antibodies (expressed as the percent of total antibody) within the pPB1750-2360 (Freunds and Gerbu) and pPB1750-2360 PEG preparations, defined quantities of these antibody preparations were chromatographed on an affinity column containing the entire toxin B repeat region (pPB1750-2360). The amount of affinity purified antibody was then quantified.[0518]

An affinity column containing the recombinant toxin B repeat protein, pPB1750-2360, was made as follows. Four ml of PBS-washed Actigel resin (Sterogene) was coupled with 5 mg of pPB1750-2360 affinity purified protein (dialyzed into PBS; estimated to be greater than 95% full length fusion protein) in a 15 ml tube (Falcon) containing {fraction (1/10)} final volume Aid-coupling solution (1M sodium cyanoborohydride). Aliquots of the supernatant from the coupling reactions, before and after coupling, were assessed by Coomassie staining of 7.5% SDS-PAGE gels. Based on protein band intensities, greater than 95% (approximately 5 mg) of recombinant protein was coupled to the resin. The coupled resin was poured into a 10 ml column (BioRad), washed extensively with PBS, pre-eluted with 4M guanidine-HCl (in 10 mM Tris-HCl, pH 8.0; 0.005% thimerosal) and re-equilibrated in PBS and stored at 4° C.[0519]

Aliquots of pMB1750-2360, pMB1750-2360 (Gerbu) or pPB1750-2360 IgY polyclonal antibody preparations (PEG preps) were affinity purified on the above column as follows. The column was attached to an UV monitor (ISCO), and washed with PBS. Forty ml aliquots of 2×PEG preps (filter sterilized using a 0.45μ filter and quantified by OD[0520]₂₈₀before chromatography) was applied. The column was washed with PBS until the baseline was re-established (the column flow-through was saved), washed with BBSTween to elute nonspecifically binding antibodies and re-equilibrated with PBS. Bound antibody was eluted from the column in 4M guanidine-HCl (in 10 mM Tris-HCL, pH 8.0, 0.005% thimerosal) and the entire elution peak collected in a 15 ml tube (Falcon). The column was re-equilibrated, and the column eluate re-chromatographed as described above. The antibody preparations were quantified by UV absorbance (the elution buffer was used to zero the spectrophotometer). Approximately 10 fold higher concentrations of total purified antibody was obtained upon elution of the first chromatography pass relative to the second pass. The low yield from the second chromatography pass indicated that most of the specific antibodies were removed by the first round of chromatography.

Pools of affinity purified specific antibodies were prepared by dialysis of the column elutes after the first column chromatography pass for the pMB1750-2360, pMB1750-2360 (Gerbu) or pPB1750-2360 IgY polyclonal antibody preparations. The elutes were collected on ice and immediately dialyzed against a 100-fold volume of. PBS at 4° C. for 2 hrs. The samples were then dialyzed against 3 changes of a 65-fold volume of PBS at 4° C. Dialysis was performed for a minimum of 8 hrs per change of PBS. The dialyzed samples were collected, centrifuged to remove insoluble debris, quantified by OD[0521]₂₈₀, and stored at 4° C.

The percentage of toxin B repeat-specific antibodies present in each preparation was determined using the quantifications of antibody yields from the first column pass (amount of specific antibody recovered after first pass/total protein loaded). The yield of repeat-specific affinity purified antibody (expressed as the percent of total protein in the preparation) in: 1) the pMB1750-2360 PEG prep was approximately 0.5%, 2) the pMB1750-2360 (Gerbu) prep was approximately 2.3%, and 3) the pPB1750-2360 prep was approximately 0.4%. Purification of a CTB IgY polyclonal antibody preparation on the same column demonstrated that the concentration of toxin B repeat specific antibodies in the CTB preparation was 0.35%.[0522]

These results demonstrate that 1) the use of Gerbu adjuvant enhanced the titer of specific antibody produced against the pMB1750-2360 protein 5-fold relative to immunization using Freunds adjuvant, and 2) the differences seen in the in vivo neutralization ability of the pMB1750-2360 (not neutralizing) and pPB1750-2360 (neutralizing) and CTB (neutralizing) PEG preps seen in Example 19 was not due to differences in the titers of repeat-specific antibodies in the three preparations because the titer of repeat-specific antibody was similar for all three preps; therefore the differing ability of the three antibody preparations to neutralize toxin B must reflect qualitative differences in the induced toxin B repeat-specific antibodies. To confirm that qualitative differences exist between antibodies raised in hens immunized with different recombinant proteins and/or different adjuvants, the same amount of affinity purified anti-toxin B repeat (aa 1870-2360 of toxin B) antibodies from the different preparations was administered to hamsters using the in vivo hamster model as described below.[0523]

b) In Vivo Neutralization of Toxin B Using Affinity Purified Antibody[0524]

The in vivo hamster model was utilized to assess the neutralizing ability of the affinity purified antibodies raised against recombinant toxin B proteins purified in (a) above. As well, a 4× IgY PEG preparation from a second independent immunization utilizing the pPB1750-2360 antigen with Freunds adjuvant was tested for in vivo neutralization. The results are shown in Table 31.[0525]

The results shown in Table 31 demonstrate that:[0526]

1) as shown in Example 19 and reproduced here, 1.5 mg of affinity purified antibody from pMB1750-2360 immunized hens using Freunds adjuvant does not neutralize toxin B in vivo. However, 300 μg of affinity purified antibody from similarly immunized hens utilizing Gerbu adjuvant demonstrated complete neutralization of toxin B in vivo. This demonstrates that Gerbu adjuvant, in addition to enhancing the titer of antibodies reactive to the pMB1750-2360 antigen relative to Freunds adjuvant (demonstrated in (a) above), also enhances the yield of neutralizing antibodies to this antigen, greater than 5 fold.[0527]

2) Complete in vivo neutralization of toxin B was observed with 1.5 mg of affinity purified antibody from hens immunized with pPB1750-2360 antigen, but not with pMB1750-2360 antigen, when Freunds adjuvant was used. This demonstrates, using standardized toxin B repeat-specific antibody concentrations, that neutralizing antibodies were induced when pPB1750-2360 but not pMB1750-2360 was used as the antigen with Freunds adjuvant.[0528]

3) Complete in vivo neutralization was observed with 300 μg of pMB1750-2360 (Gerbu) antibody, but not with 300 μg of pPB1750-2360 (Freunds) antibody. Thus the pMB1750-2360 (Gerbu) antibody has a higher titer of neutralizing antibodies than the pPB1750-2360 (Freunds) antibody.[0529]

4) Complete neutralization of toxin B was observed using 300 μg of CTB antibody [affinity purified (AP)] but not 100 μg CTB antibody (AP or PEG prep). This demonstrates that greater than 100 μg of toxin B repeat-specific antibody (anti-CTB) is necessary to neutralize 25 μg toxin B in vivo in this assay, and that affinity purified antibodies specific to the toxin B repeat interval neutralize toxin B as effectively as the PEP prep of IgY raised against the entire CTB protein (shown in this assay).[0530]

5) As was observed with the initial pPB1750-2360 (IgY) PEG preparation (Example 19), complete neutralization was observed with a IgY PEG preparation isolated from a second independent group of pPB1750-2360 (Freunds) immunized hens. This demonstrates that neutralizing antibodies are reproducibly produced when hens are immunized with pPB1750-2360 protein utilizing Freunds adjuvant.[0531]

TABLE 31


In vivo Neutralization Of Toxin B Using Affinity Purified Antibodies

	Number	Number
Treatment Group^a	Animals Alive^b	Animals Dead^b

Preimmune¹	0	5
CTB (300 μg)²	5	0
CTB (100 μg)²	1	4
pMB1750-2360 (G) (5 mg)²	5	0
pMB1750-2360 (G) (1.5 mg)²	5	0
pMB1750-2360 (G) (300 μg)²	5	0
pMB1750-2360 (F) (1.5 mg)²	0	5
pPB1750-2360 (F) (1.5 mg)²	5	0
pPB1750-2360 (F) (300 μg)²	1	4
CTB (100 μg)³	2	3
pPB1750-2360 (F) (500 μg)¹	5	0

Example 21Diagnostic Enzyme Immunoassays forC. difficileToxins A and B

The ability of the recombinant toxin proteins and antibodies raised against these recombinant proteins (described in the above examples) to form the basis of diagnostic assays for the detection of clostridial toxin in a sample was examined. Two immunoassay formats were tested to quantitatively detect[0532]C. difficiletoxin A and toxin B from a biological specimen. The first format involved a competitive assay in which a fixed amount of recombinant toxin A or B was immobilized on a solid support (e.g., microtiter plate wells) followed by the addition of a toxin-containing biological specimen mixed with affinity-purified or PEG fractionated antibodies against recombinant toxin A or B. If toxin is present in a specimen, this toxin will compete with the immobilized recombinant toxin protein for binding to the anti-recombinant antibody thereby reducing the signal obtained following the addition of a reporter reagent. The reporter reagent detects the presence of antibody bound to the immobilized toxin protein.

In the second format, a sandwich immunoassay was developed using affinity-purified antibodies to recombinant toxin A and B. The affinity-purified antibodies to recombinant toxin A and B were used to coat microtiter wells instead of the recombinant polypeptides (as was done in the competitive assay format). Biological samples containing toxin A or B were then added to the wells followed by the addition of a reporter reagent to detect the presence of bound toxin in the well.[0533]

a) Competitive Immunoassay for the Detection of[0534]C. difficileToxin

Recombinant toxin A or B was attached to a solid support by coating 96 well microtiter plates with the toxin protein at a concentration of 1 μg/ml in PBS. The plates were incubated overnight at 2-8° C. The following morning, the coating solutions were removed and the remaining protein binding sites on the wells were blocked by filling each well with a PBS solution containing 0.5% BSA and 0.05% Tween-20. Native[0535]C. difficiletoxin A or B (Tech Lab) was diluted to 4 μg/ml in stool extracts from healthy Syrian hamsters (Sasco). The stool extracts were made by placing fecal pellets in a 15 ml centrifuge tube; PBS was added at 2 ml/pellet and the tube was vortexed to create a uniform suspension. The tube was then centrifuged at 2000 rpm for 5 min at room temperature. The supernatant was removed; this comprises the stool extract. Fifty μl of the hamster stool extract was pipetted into each well of the microtiter plates to serve as the diluent for serial dilutions of the 4 μg/ml toxin samples. One hundred μl of the toxin samples at 4 μg/ml was pipetted into the first row of wells in the microtiter plate, and 50 μl aliquots were removed and diluted serially down the plate in duplicate. An equal volume of affinity purified anti-recombinant toxin antibodies [1 ng/well of anti-pMA1870-2680 antibody was used for the detection of toxin A; 0.5 ng/well of anti-pMB1750-2360(Gerbu) was used for the detection of toxin B] were added to appropriate wells, and the plates were incubated at room temperature for 2 hours with gentle agitation. Wells serving as negative control contained antibody but no native toxin to compete for binding.

Unbound toxin and antibody were removed by washing the[0536]plates 3 to 5 times with PBS containing 0.05% Tween-20. Following the wash step, 100 μl of rabbit anti-chicken IgG antibody conjugated to alkaline phosphatase (Sigma) was added to each well and the plates were incubated for 2 hours at room temperature. The plates were then washed as before to remove unbound secondary antibody. Freshly prepared alkaline phosphatase substrate (1 mg/ml p-nitrophenyl phosphate (Sigma) in 50 mM Na₂CO₃, pH 9.5; 10 mM MgCl₂) was added to each well. Once sufficient color developed, the plates were read on a Dynatech MR700 microtiter plate reader using a 410 nm filter.

The results are summarized in Tables 32 and 33. For the results shown in Table 32, the wells were coated with recombinant toxin A protein (PMA1870-2680). The amount of native toxin A added (present as an addition to solubilized hamster stool) to a given well is indicated (0 to 200 ng). Antibody raised against the recombinant toxin A protein, pMA1870-2680, was affinity purified on the an affinity column containing pPA1870-2680 (described in Example 20). As shown in Table 32, the recombinant toxin A protein and affinity-purified antitoxin can be used for the basis of a competitive immunoassay for the detection of toxin A in biological samples.[0537]

In this competition assay, the reduction is considered significant over the background levels at all points; therefore the assay can be used to detect samples containing less than 12.5 ng toxin A/well and as little as 50-100 ng toxin B/well.[0539]

TABLE 32


Competitive Inhibition Of Anti-C.difficile
Toxin A By Native Toxin A

	ng Toxin A/Well	OD₄₁₀Readout

	200	0.176
	100	0.253
	50	0.240
	25	0.259
	12.5	0.309
	6.25	0.367
	3.125	0.417
	0	0.590

[0540]

TABLE 33


Competitive Inhibition Of Anti-C.difficile
Toxin B By Native Toxin B

	ng Toxin B/Well	OD₄₁₀Readout

	200	0.392
	100	0.566
	50	0.607
	25	0.778
	12.5	0.970
	6.25	0.902
	3.125	1.040
	0	1.055

These competitive inhibition assays demonstrate that native[0541]C. difficiletoxins and recombinantC. difficiletoxin proteins can compete for binding to antibodies raised against recombinantC. difficiletoxins demonstrating that these anti-recombinant toxin antibodies provide effective diagnostic reagents.

b) Sandwhich Immunoassay for the Detection of[0542]C. difficileToxin

Affinity-purified antibodies against recombinant toxin A or toxin B were immobilized to 96 well microtiter plates as follows. The wells were passively coated overnight at 4° C. with affinity purified antibodies raised against either pMA1870-2680 (toxin A) or pMB1750-2360 (Gerbu) (toxin B). The antibodies were affinity purified as described in Example 20. The antibodies were used at a concentration of 1 μg/ml and 100 μl was added to each microtiter well. The wells were then blocked with 200 μl of 0.5% BSA in PBS for 2 hours at room temperature and the blocking solution was then decanted. Stool samples from healthy Syrian hamsters were resuspended in PBS, pH 7.4 (2 ml PBS/stool pellet was used to resuspend the pellets and the sample was centrifuged as described above). The stool suspension was then spiked with native[0543]C. difficile toxin A or B (Tech Lab) at4 μg/ml. The stool suspensions containing toxin (either toxin A or toxin B) were then serially diluted two-fold in stool suspension without toxin and 50 μl was added in duplicate to the coated microtiter wells. Wells containing stool suspension without toxin served as the negative control.

The plates were incubated for 2 hours at room temperature and then were washed three times with PBS. One hundred μl of either goat anti-native toxin A or goat anti-native toxin B (Tech Lab) diluted 1:1000 in PBS containing 1% BSA and 0.05[0544]% Tween 20 was added to each well. The plates were incubated for another 2 hours at room temperature. The plates were then washed as before and 1001 μl of alkaline phosphatase-conjugated rabbit anti-goat IgG (Cappel, Durham, N.C.) was added at a dilution of 1:1000. The plates were incubated for another 2 hours at room temperature. The plates were washed as before then developed by the addition of 100 μl/well of a substrate solution containing 1 mg/ml p-nitrophenyl phosphate (Sigma) in 50 mM Na₂CO₃, pH 9.5; 10 mM MgCl₂. The absorbance of each well was measured using a plate reader (Dynatech) at 410 nm. The assay results are shown in Tables 34 and 35.

TABLE 34


C. difficileToxin A Detection In Stool Using
Affinity-Purified Antibodies Against Toxin A

	ng Toxin A/Well	OD₄₁₀Readout

	200	0.9
	100	0.8
	50	0.73
	25	0.71
	12.5	0.59
	6.25	0.421
	0	0

[0545]

TABLE 35


C. difficileToxin B Detection In Stool Using
Affinity-Purified Antibodies Against Toxin B

	ng Toxin B/Well	OD₄₁₀Readout

	200	1.2
	100	0.973
	50	0.887
	25	0.846
	12.5	0.651
	6.25	0.431
	0	0.004

The results shown in Tables 34 and 35 show that antibodies raised against recombinant toxin A and toxin B fragments can be used to detect the presence of[0546]C. difficiletoxin in stool samples. These antibodies form the basis for a sensitive sandwich immunoassay which is capable of detecting as little as 6.25 ng of either toxin A or B in a 50 μl stool sample. As shown above in Tables 34 and 35, the background for this sandwich immunoassay is extremely low; therefore, the sensitivity of this assay is much lower than 6.25 ng toxin/well. It is likely that toxin levels of 0.5 to 1.0 pg/well could be detected by this assay.

The results shown above in Tables 32-35 demonstrate clear utility of the recombinant reagents in[0547]C. difficiletoxin detection systems.

Example 22Construction and Expression ofC. botulinumC Fragment Fusion Proteins

The[0548]C. botulinumtype A neurotoxin gene has been cloned and sequenced [Thompson, et al., Eur. J. Biochem. 189:73 (1990)]. The nucleotide sequence of the toxin gene is available from the EMBL/GenBank sequence data banks under the accession number X52066; the nucleotide sequence of the coding region is listed in SEQ ID NO:27. The amino acid sequence of theC. botulinumtype A neurotoxin is listed in SEQ ID NO:28. The type A neurotoxin gene is synthesized as a single polypeptide chain which is processed to form a dimer composed of a light and a heavy chain linked via disulfide bonds. The 50 kD carboxy-terminal portion of the heavy chain is referred to as the C fragment or the H_Cdomain.

Previous attempts by others to express polypeptides comprising the C fragment of[0549]C. botulinumtype A toxin as a native polypeptide (e.g., not as a fusion protein) inE. colihave been unsuccessful [H. F. LaPenotiere, et al. inBotulinum and Tetanus Neurotoxins, DasGupta, Ed., Plenum Press, New York (1993), pp. 463-466]. Expression of the C fragment as a fusion with theE. coliMBP was reported to result in the production of insoluble protein (H. F. LaPenotiere, et al., supra).

In order to produce soluble recombinant C fragment proteins in[0550]E. coli, fusion proteins comprising a synthetic C fragment gene derived from theC. botulinumtype A toxin and either a portion of theC. difficiletoxin protein or the MBP were constructed. This example involved a) the construction of plasmids encoding C fragment fusion proteins and b) expression ofC. botulinumC fragment fusion proteins inE. coli.

a) Construction of Plasmids Encoding C Fragment Fusion Proteins[0551]

In Example 11, it was demonstrated that the[0552]C difficiletoxin A repeat domain can be efficiently expressed and purified inE. colias either native (expressed in the pET 23a vector in clone pPA1870-2680) or fusion (expressed in the pMALc vector as a fusion with theE. coliMBP in clone pMA1870-2680) proteins. Fusion proteins comprising a fusion between the MBP, portions of theC. difficiletoxin A repeat domain (shown to be expressed as a soluble fusion protein) and the C fragment of theC. botulinumtype A toxin were constructed. A fusion protein comprising the C fragment of theC. botulinumtype A toxin and the MBP was also constructed.

FIG. 25 provides a schematic representation of the botulinal fusion proteins along with the donor constructs containing the[0553]C. difficiletoxin A sequences orC. botulinumC fragment sequences which were used to generate the botulinal fusion proteins. In FIG. 25, the solid boxes representC. difficiletoxin A gene sequences, the open boxes representC. botulinumC fragment sequences and the solid black ovals represent theE. coliMBP. When the name for a restriction enzyme appears inside parenthesis, this indicates that the restriction site was destroyed during construction. An asterisk appearing with the name for a restriction enzyme indicates that this restriction site was recreated at the cloning junction.

In FIG. 25, a restriction map of the pMA1870-2680 and pPA1100-2680 constructs (described in Example 11) which contain sequences derived from the[0554]C. difficiletoxin A repeat domain are shown; these constructs were used as the source ofC. difficiletoxin A gene sequences for the construction of plasmids encoding fusions between theC. botulinumC fragment gene and theC. difficiletoxin A gene. The pMA1870-2680 expression construct expresses high levels of soluble, intact fusion protein (20 mg/liter culture) which can be affinity purified on an amylose column (purification described in Example 11d).

The pAlterBot construct (FIG. 25) was used as the source of[0555]C. botulinumC fragment gene sequences for the botulinal fusion proteins. pAlterBot was obtained from J. Middlebrook and R. Lemley at the U.S. Department of Defense. pAlterBot contains a syntheticC. botulinumC fragment inserted in to the pALTER-1® vector (Promega). This synthetic C fragment gene encodes the same amino acids as does the naturally occurring C fragment gene. The naturally occurring C fragment sequences, like most clostridial genes, are extremely A/T rich (Thompson et al., supra). This high A/T content creates expression difficulties inE. coliand yeast due to altered codon usage frequency and fortuitous polyadenylation sites, respectively. In order to improve the expression of C fragment proteins inE. coli, a synthetic version of the gene was created in which the non-preferred codons were replaced with preferred codons.

The nucleotide sequence of the[0556]C. botulinumC fragment gene sequences contained within pAlterBot is listed in SEQ ID NO:22. The first six nucleotides (ATGGCT) encode a methionine and alanine residue, respectively. These two amino acids result from the insertion of theC. botulinumC fragment sequences into the pALTER® vector and provide the initiator methionine residue. The amino acid sequence of theC. botulinumC fragment encoded by the sequences contained within pAlterBot is listed in SEQ ID NO:23. The first two amino acids (Met Ala) are encoded by vector-derived sequences. From the third amino acid residue onward (Arg), the amino acid sequence is identical to that found in theC. botulinumtype A toxin gene.

The pMA1870-2680, pPA1100-2680 and pAlterBot constructs were used as progenitor plasmids to make expression constructs in which fragm ents of the[0557]C. difficiletoxin A repeat domain were expressed as genetic fusions with theC. botulinumC fragment gene using the pMAL-c expression vector (New England BioLabs). The pMAL-c expression vector generates fusion proteins which contain the MBP at the amino-terminal end of the protein. A construct, pMBot, in which theC. botulinumC fragment gene was expressed as a fusion with only the MBP was constructed (FIG. 25). Fusion protein expression was induced fromE. colistrains harboring the above plasmids, and induced protein was affinity purified on an amylose resin column.

i) Construction of pBlueBot[0558]

In order to facilitate the cloning of the[0559]C. botulinumC fragment gene sequences into a number of desired constructs, the botulinal gene sequences were removed from pAlterBot and were inserted into the pBluescript plasmid (Stratagene) to generate pBlueBot (FIG. 25). pBlueBot was constructed as follows. Bacteria containing the pAlterBot plasmid were grown in medium containing tetracycline and plasmid DNA was isolated using the QIAprep-spin Plasmid Kit (Qiagen). One microgram of pAlterBot DNA was digested with NcoI and the resulting 3′ recessed sticky end was made blunt using the Klenow fragment of DNA polymerase I (here after the Klenow fragment). The pAlterBot DNA was then digested with HindIII to release the botulinal gene sequences (the Bot insert) as a blunt (filled NcoI site)-HindIII fragment. pBluescript vector DNA was prepared by digesting 200 ng of pBluescript DNA with SmaI and HindIII. The digestion products from both plasmids were resolved on an agarose gel. The appropriate fragments were removed from the gel, mixed and purified utilizing the Prep-a-Gene kit (BioRad). The eluted DNA was then ligated using T4 DNA ligase and used to transform competent DH5a cells (Gibco-BRL). Host cells were made competent for transformation using the calcium chloride protocol of Sambrook et al., supra at 1.82-1.83. Recombinant clones were isolated and confirmed by restriction digestion using standard recombinant molecular biology techniques (Sambrook et al, supra). The resultant clone, pBlueBot, contains several useful unique restriction sites flanking the Bot insert (i.e., theC. botulinumC fragment sequences derived from pAlterBot) as shown in FIG. 25.

ii) Construction of[0560]C. difficile/C. botulinum/MBP Fusion Proteins

Constructs encoding fusions between the[0561]C. difficiletoxin A gene and theC. botulinumC fragment gene and the MBP were made utilizing the same recombinant DNA methodology outlined above; these fusion proteins contained varying amounts of theC. difficiletoxin A repeat domain.

The pMABot clone contains a 2.4 kb insert derived from the[0562]C. difficiletoxin A gene fused to the Bot insert (i.e, theC. botulinumC fragment sequences derived from pAlterBot). pMABot (FIG. 25) was constructed by mixing gel-purified DNA from NotI/HindIII digested pBlueBot (the 1.2 kb Bot fragment), SpeI/NotI digested pPA1100-2680 (the 2.4 kbC. difficiletoxin A repeat fragment) and XbaI/HindIII digested pMAL-c vector. Recombinant clones were isolated, confirmed by restriction digestion and purified using the QIAprep-spin Plasmid Kit (Qiagen). This clone expresses the toxin A repeats and the botulinal C fragment protein sequences as an in-frame fusion with the MBP.

The pMCABot construct contains a 1.0 kb insert derived from the[0563]C. difficiletoxin A gene fused to the Bot insert (i.e, theC. botulinumC fragment sequences derived from pAlterBot). pMCABot was constructed by digesting the pMABot clone with EcoRI to remove the 5′ end of theC. difficiletoxin A repeat (see FIG. 25, the pMAL-c vector contains aEcoRI site 5′ to theC. difficileinsert in the pMABot clone). The restriction sites were filled and religated together after gel purification. The resultant clone (pMCABot, FIG. 25) generated an in-frame fusion between the MBP and the remaining 3′ portion of theC. difficiletoxin A repeat domain fused to the Bot gene.

The pMNABot clone contains the 1 kb SpeI/EcoRI (filled) fragment from the[0564]C. difficiletoxin A repeat domain (derived from clone pPA 1100-2680) and the 1.2 kbC. botulinumC fragment gene as a NcoI (filled)/HindIII fragment (derived from pAlterBot). These two fragments were inserted into the pMAL-c vector digested with XbaI/HindIII. The two insert fragments were generated by digestion of the appropriate plasmid with EcoRI (pPAI 100-2680) or. NcoI (pAlterBot) followed by treatment with the Klenow fragment. After treatment with the Klenow fragment, the plasmids were digested with the second enzyme (either SpeI or HindIII). All three fragments were gel purified, mixed and Prep-a-Gene purified prior to ligation. Following ligation and transformation, putative recombinants were analyzed by restriction analysis; the EcoRI site was found to be regenerated at the fusion junction, as was predicted for a fusion between the filled EcoRI and NcoI sites.

A construct encoding a fusion protein between the botulinal C fragment gene and the MBP gene was constructed (i.e., this fusion lacks any[0565]C. difficiletoxin A gene sequences) and termed pMBot. The pMBot construct was made by removal of theC. difficiletoxin A sequences from the pMABot construct and fusing the C fragment gene sequences to the MBP. This was accomplished by digestion of pMABot DNA with StuI (located in thepMALc polylinker 5′ to the XbaI site) and XbaI (located 3′ to the NotI site at the toxA-Bot fusion junction), filling in the XbaI site using the Klenow fragment, gel purifying the desired restriction fragment, and ligating the blunt ends to circularize the plasmid. Following ligation and transformation, putative recombinants were analyzed by restriction mapping of the Bot insert (i.e, theC. botulinumC fragment sequences).

b) Expression of[0566]C. botulinumC Fragment Fusion Proteins inE. coli

Large scale (1 liter) cultures of the pMAL-c vector, and each recombinant construct described above in (a) were grown, induced, and soluble protein fractions were isolated as described in Example 18. The soluble protein extracts were chromatographed on amylose affinity columns to isolate recombinant fusion protein. The purified recombinant fusion proteins were analyzed by running samples on SDS-PAGE gels followed by Coomassie staining and by Western blot analysis as described [Williams et al, (1994) supra]. In brief, extracts were prepared and chromatographed in column buffer (10 mM NaPO[0567]₄, 0.5 M NaCl, 10 mM β-mercaptoethanol, pH 7.2) over an amylose resin (New England Biolabs) column, and eluted with column buffer containing 10 mM maltose as described [Williams, et al. (1994), supra]. An SDS-PAGE gel containing the purified protein samples stained with Coomassie blue is shown in FIG. 26.

In FIG. 26, the following samples were loaded. Lanes 1-6 contain protein purified from[0568]E. colicontaining the pMAL-c, pPA1870-2680, pMABot, pMNABot, pMCABot and pMBot plasmids, respectively.Lane 7 contains broad range molecular weight protein markers (BioRad).

The protein samples were prepared for electrophoresis by mixing 5 μl of eluted protein with 5 μl of 2×SDS-PAGE sample buffer (0.125 mM Tris-HCl, pH 6.8, 2 mM EDTA, 6% SDS, 20% glycerol, 0.025% bromophenol blue; β-mercaptoethanol is added to 5% before use). The samples were heated to 95° C. for 5 min, then cooled and loaded on a 7.5% agarose SDS-PAGE gel. Broad range molecular weight protein markers were also loaded to allow estimation of the MW of identified fusion proteins. After electrophoresis, protein was detected generally by staining the gel with Coomassie blue.[0569]

In all cases the yields were in excess of 20 mg fusion protein per liter culture (see Table 36) and, with the exception of the pMCABot protein, a high percentage (i.e., greater than 20-50% of total eluted protein) of the eluted fusion protein was of a MW predicted for the full length fusion protein (FIG. 26). It was estimated (by visual inspection) that less than 10% of the pMCABot fusion protein was expressed as the full length fusion protein.[0570]

TABLE 36


Yield Of Affinity PurifiedC. botulinum
C Fragment/MBP Fusion Proteins

	Yield	Percentage Of Total
Construct	(mg/liter of Culture)	Soluble Protein

pMABot	24	5.0
pMCABot	34	5.0
pMNABot	40	5.5
pMBot	22	5.0
pMA1870-2680	40	4.8

These results demonstrate that high level expression of intact[0571]C. botulinumC fragment/C. difficiletoxin A fusion proteins inE. coliis feasible using the pMAL-c expression system. These results are in contrast to those reported by H. F. LaPenotiere, et al. (1993), supra. In addition, these results show that it is not necessary to fuse the botulinal C fragment gene to theC. difficiletoxin A gene in order to produce a soluble fusion protein using the pMAL-c system inE. coli.

In order to determine whether the above-described botulinal fusion proteins were recognized by anti-[0572]C. botulinumtoxin A antibodies, Western blots were performed. Samples containing affinity-purified proteins fromE. colicontaining the pMABot, pMCABot, pMNABot, pMBot, pMA1870-2680 or pMALc plasmids were analyzed. SDS-PAGE gels (7.5% acrylamide) were loaded with protein samples purified from each expression construct. After electrophoresis, the gels were blotted and protein transfer was confirmed by Ponceau S staining (as described in Example 12b).

Following protein transfer, the blots were blocked by incubation for 1 hr at 20C in blocking buffer [PBST (PBS containing 0.1[0573]

% Tween

20 and 5% dry milk)]. The blots were then incubated in 10 ml of a solution containing the primary antibody; this solution comprised a 1/50 dilution of an anti-C. botulinumtoxin A IgY PEG prep (described in Example 3) in blocking buffer. The blots were incubated for 1 hr at room temperature in the presence of the primary antibody. The blots were washed and developed using a rabbit anti-chicken alkaline phosphatase conjugate (Boehringer Mannheim) as the secondary antibody as follows. The rabbit anti-chicken antibody was diluted to 1 μg/ml in blocking buffer (10 ml final volume per blot) and the blots were incubated at room temperature for 1 hour in the presence of the secondary antibody. The blots were then washed successively with PBST, BBS-Tween and 50 mM Na₂CO₃, pH 9.5. The blots were then developed in freshly-prepared alkaline phosphatase substrate buffer (100 μg/ml nitro blue tetrazolium, 50 μg/ml 5-bromo-chloro-indolylphosphate, 5 mM MgCl₂in 50 mM Na2CO₃, pH 9.5). Development was stopped by flooding the blots with distilled water and the blots were air dried.

This Western blot analysis detected anti-[0574]C. botulinumtoxin reactive proteins in the pMABot, pMCABot, pMNABot and pMBot protein samples (corresponding to the predicted full length proteins identified above by Coomassie staining in FIG. 26), but not in the pMA1100-2680 or pMALc protein samples.

These results demonstrate that the relevant fusion proteins purified on an amylose resin as described above in section a) contained immunoreactive[0575]C. botulinumC fragment protein as predicted.

Example 23Generation of Neutralizing Antibodies by Nasal Administration of pMBot Protein

The ability of the recombinant botulinal toxin proteins produced in Example 22 to stimulate a systemic immune response against botulinal toxin epitopes was assessed. This example involved: a) the evaluation of the induction of serum IgG titers produced by nasal or oral administration of botulinal toxin-containing[0576]C. difficiletoxin A fusion proteins and b) the in vivo neutralization ofC. botulinumtype A neurotoxin by anti-recombinantC. botulinumC fragment antibodies.

a) Evaluation of the Induction of Serum IgG Titers Produced by Nasal or Oral Administration of Botulinal Toxin-Containing[0577]C. difficileToxin A Fusion Proteins

Six groups containing five 6 week old CF female rats (Charles River) per group were immunized nasally or orally with one of the following three combinations using protein prepared in Example 22: (1) 250 μg pMBot protein per rat (nasal and oral); 2) 250 μg pMABot protein per rat (nasal and oral); 3) 125 μg pMBot admixed with 125 μg pMA1870-2680 per rat (nasal and oral). A second set of 5 groups containing 3 CF female rats/group were immunized nasally or orally with one of the following combinations (4) 250 μg pMNABot protein per rat (nasal and oral) or 5) 250 μg pMAL-c protein per rat (nasal and oral).[0578]

The fusion proteins were prepared for immunization as follows. The proteins (in column buffer containing 10 mM maltose) were diluted in 0.1 M carbonate buffer, pH 9.5 and administered orally or nasally in a 200 μl volume. The rats were lightly sedated with ether prior to administration. The oral dosing was accomplished using a 20 gauge feeding needle. The nasal dosing was performed using a P-200 micro-pipettor (Gilson). The rats were boosted 14 days after the primary immunization using the techniques described above and were bled 7 days later. Rats from each group were lightly etherized and bled from the tail. The blood was allowed to clot at 37° C. for 1 hr and the serum was collected.[0579]

The serum from individual rats was analyzed using an ELISA to determine the anti-[0580]C. botulinumtype A toxin IgG serum titer. The ELISA protocol used is a modification of that described in Example 13c. Briefly, 96-well microtiter plates (Falcon, Pro-Bind Assay Plates) were coated withC. botulinumtype A toxoid (prepared as described in Example 3a) by placing 100 μl volumes ofC. botulinumtype A toxoid at 2.5 μg/ml in PBS containing 0.005% thimerosal in each well and incubating overnight at 4° C. The next morning, the coating suspensions were decanted and all wells were washed three times using PBS.

In order to block non-specific binding sites, 100 μl of blocking solution [0.5% BSA in PBS] was then added to each well and the plates were incubated for 1 hr at 37° C. The blocking solution was decanted and duplicate samples of 150 μl of diluted rat serum added to the first well of a dilution series. The initial testing serum dilution was 1:30 in blocking solution containing 0.5[0581]% Tween 20 followed by 5-fold dilutions into this solution. This was accomplished by serially transferring 30 μl aliquots to 120 μl blocking solution containing 0.5% Tween 20, mixing, and repeating the dilution into a fresh well. After the final dilution, 30 μl was removed from the well such that all wells contained 120 μl final volume. A total of 3 such dilutions were performed (4 wells total). The plates were incubated 1 hr at 37° C. Following this incubation, the serially diluted samples were decanted and the wells were washed six times using PBS containing 0.5% Tween 20 (PBST). To each well, 100 μl of a rabbit anti-Rat IgG alkaline phosphatase (Sigma) diluted (1/1000) in blocking buffer containing 0.5% Tween 20 was added and the plate was incubated for 1 hr at 37° C. The conjugate solutions were decanted and the plates were washed as described above, substituting 50 mM Na₂CO₃, pH 9.5 for the PBST in the final wash. The plates were developed by the addition of −100 μl of a solution containing 1 mg/ml para-nitro phenyl phosphate (Sigma) dissolved in 50 mM Na₂CO₃, 10 mM MgCl₂, pH 9.5 to each well, and incubating the plates at room temperature in the dark for 5-45 min. The absorbency of each well was measured at 410 nm using a Dynatech MR 700 plate reader. The results are summarized in Tables 37 and 38 and represent mean serum reactivities of individual mice.

TABLE 37


Determination Of Anti-C. botulinum Type A Toxin Serum IgG Titers Following Immunization
WithC. botulinum C Fragment-Containing Fusion
Proteins

Nasal

Oral

Route of Immunization

pMBot &

pMBot&

	PRE-		pMA1870-			pMA1870-
Immunogen	IMMUNE	pMBot	2680	pMABot	pMBot	2680	pMABot

Dilution
1:30	0.080	1.040	1.030	0.060	0.190	0.080	0.120
1:150	0.017	0.580	0.540	* 0.022	0.070	0.020	0.027
1:750	0.009	0.280	0.260	0.010	0.020	0.010	0.014
1:3750	0.007	0.084	0.090	0.009	0.009	0.010	0.007
# Rats		5	5	5	5	2	2
Tested

[0582]

TABLE 38


Determination Of Anti-C. botulinum Type A Toxin Serum IgG Titers
Following Immunization WithC. botulinum C
Fragment-Containing Fusion Proteins

Route of Immunization

PRE-

Nasal

Oral

Immunogen	IMMUNE	pMBot	pMABot	pMNABot	pMNABot

Dilution

1:30	0.040	0.557	0.010	0.015	0.010
1:150	0.009	0.383	0.001	0.003	0.002
1:750	0.001	0.140	0.000	0.000	0.000
1:3750	0.000	0.040	0.000	0.000	0.000
# Rats		1	1	3	3
Tested

The above ELISA results demonstrate that reactivity against the botulinal fusion proteins was strongest when the route of administration was nasal; only weak responses were stimulated when the botulinal fusion proteins were given orally. Nasally delivered pMbot and pMBot admixed with pMA1870-2680 invoked the greatest serum IgG response. These results show that only the pMBot protein is necessary to induce this response, since the addition of the pMA1870-2680 protein did not enhance antibody response (Table 37). Placement of the[0583]C. difficiletoxin A fragment between the MBP and theC. botulinumC fragment protein dramatically reduced anti-bot IgG titer (see results using pMABot, pMCABot and pMNABot proteins).

This study demonstrates that the pMBot protein induces a strong serum IgG response directed against[0584]C. botulinumtype A toxin when nasally administered.

b) In Vivo Neutralization of[0585]C. botulinumType A Neurotoxin by Anti-RecombinantC. botulinumC Fragment Antibodies

The ability of the anti-[0586]C. botulinumtype A toxin antibodies generated by nasal administration of recombinant botulinal fusion proteins in rats (Example 22) to neutralizeC. botulinumtype A toxin was tested in a mouse neutralization model. The mouse model is the art accepted method for detection of botulinal toxins in body fluids and for the evaluation of anti-botulinal antibodies [E. J. Schantz and D. A. Kautter, J. Assoc. Off. Anal. Chem. 61:96 (1990) and Investigational New Drug (BB-IND-3703) application by the Surgeon General of the Department of the Army to the Federal Food and Drug Administration]. The anti-C. botulinumtype A toxin antibodies were prepared as follows.

Rats from the group given pMBot protein by nasal administration were boosted a second time with 250 μg pMBot protein per rat and serum was collected 7 days later. Serum from one rat from this group and from a preimmune rat was tested for anti-[0587]C. botulinumtype A toxin neutralizing activity in the mouse neutralization model described below.

The LD[0588]₅₀of a solution of purifiedC. botulinumtype A toxin complex, obtained from Dr. Eric Johnson (University of Wisconsin Madison), was determined using the intraperitoneal (IP) method of Schantz and Kautter [J. Assoc. Off. Anal. Chem. 61:96 (1978)] using 18-22 gram female ICR mice and was found to be 3500 LD₅₀/ml. The determination of the LD₅₀was performed as follows. A Type A toxin standard was prepared by dissolving purified type A toxin complex in 25 mM sodium phosphate buffer, pH 6.8 to yield a stock toxin solution of 3.15×10⁷LD₅₀/mg. The OD₂₇₈of the solution was determined and the concentration was adjusted to 10-20 μg/ml. The toxin solution was then diluted 1:100 in gel-phosphate (30 mM phosphate, pH 6.4; 0.2% gelatin). Further dilutions of the toxin solution were made as shown below in Table 39. Two mice were injected IP with 0.5 ml of each dilution shown and the mice were observed for symptoms of botulism for a period of 72 hours.

TABLE 39


Determination Of The LD₅₀Of Purified
C. botulinumType A Toxin Complex

	Dilution	Number Dead At 72 hr

	1:320	2/2
	1:640	2/2
	1:1280	2/2
	1:2560	0/2 (sick after 72 hr)
	1:5120	0/2 (no symptoms)

From the results shown in Table. 39, the toxin titer was assumed to be between 2560 LD[0589]₅₀/ml and 5120 LD₅₀/ml (or about 3840 LD₅₀ml). This value was rounded to 3500 LD₅₀ml for the sake of calculation.

The amount of neutralizing antibodies present in the serum of rats immunized nasally with pMBot protein was then determined. Serum from two rats boosted with pMBot protein as described above and preimmune serum from one rat was tested as follows. The toxin standard was diluted 1:100 in gel-phosphate to a final concentration of 350 LD[0590]₅₀/ml. One milliliter of the diluted toxin standard was mixed with 25 μl of serum from each of the three rats and 0.2 ml of gel-phosphate. The mixtures were incubated at room temperature for 30 min with occasional mixing. Each of two mice were injected with IP with 0.5 ml of the mixtures. The mice were observed for signs of botulism for 72 hr. Mice receiving serum from rats immunized with pMBot protein neutralized this challenge dose. Mice receiving preimmune rat serum died in less than 24 hr.

The amount of neutralizing anti-toxin antibodies present in the serum of rats immunized with pMBot protein was then quantitated. Serum antibody titrations were performed by mixing 0.1 ml of each of the antibody dilutions (see Table 40) with 0.1 ml of a 1:10 dilution of stock toxin solution (3.5×10[0591]⁴LD₅₀/ml) with 1.0 ml of gel-phosphate and injecting 0.5 ml IP into 2 mice per dilution. The mice were then observed for signs of botulism for 3 days (72 hr). The results are tabulated in Table 39.

As shown in Table 40 pMBot serum neutralized[0592]C. botulinumtype A toxin complex when used at a dilution of 1:320 or less. A mean neutralizing value of 168 IU/ml was obtained for the pMBot serum (an IU is defined as 10,000 mouse LD₅₀). This value translates to a circulating serum titer of about 3.7 IU/mg of serum protein. This neutralizing titer is comparable to the commercially available bottled concentrated (Connaught Laboratories, Ltd.) horse anti-C. botulinumantiserum. A 10 ml vial of Connaught antiserum contains about 200 mg/ml of protein;each ml can neutralize 750 IU ofC. botulinumtype A toxin. After administration of one vial to a human, the circulating serum titer of the Connaught preparation would be approximately 25 lU/ml assuming an average serum volume of 3 liters). Thus, the circulating anti-C botulinum titer seen in rats nasally immunized with pMBot protein (168 IU/ml) is 6.7 time higher than the necessary circulation titer of anti-C. botulinumantibody needed to be protective in humans.

TABLE 40


Quantitation Of Neutralizing Antibodies In pMBot Sera

pMBot^a

Dilution	Rat 1	Rat 2

1:20	2/2	2/2
1:40	2/2	2/2
1:80	2/2	2/2
1:160	2/2	2/2
1:320	2/2^b	2/2^b
1:640	0/2	0/2
1:1280	0/2	0/2
1:2560	0/2	0/2

These results demonstrate that antibodies capable of neutralizing[0593]C. botulinumtype A toxin are induced when recombinantC. botulinumC fragment fusion protein produced inE. coliis used as an immunogen.

Example 24Production of SolubleC. botulinumC Fragment Protein Substantially Free of Endotoxin Contamination

Example 23 demonstrated that neutralizing antibodies are generated by immunization with the pMBot protein expressed in[0594]E. coli. These results showed that the pMBot fusion protein is a good vaccine candidate. However, immunogens suitable for use as vaccines should be pyrogen-free in addition to having the capability of inducing neutralizing antibodies. Expression clones and conditions that facilitate the production ofC. botulinumC fragment protein for utililization as a vaccine were developed.

The example involved: (a) determination of pyrogen content of the pMBot protein; (b) generation of[0595]C. botulinumC fragment protein free of the MBP; (c) expression ofC. botulinumC fragment protein using various expression vectors; and (d) purification of solubleC. botulinumC fragment protein substantially free of significant endotoxin contamination.

a) Determination of the Pyrogen Content of the pMBot Protein[0596]

In order to use a recombinant antigen as a vaccine in humans or other animals, the antigen preparation must be shown to be free of pyrogens. The most significant pyrogen present in preparations of recombinant proteins produced in gram-negative bacteria, such as[0597]E. coli, is endotoxin [F. C. Pearson, Pyrogens: endotoxins, LAL testing and depyrogentaion, (1985) Marcel Dekker, New York, pp. 23-56]. To evaluate the utility of the pMBot protein as a vaccine candidate, the endotoxin content in MBP fusion proteins was determined.

The endotoxin content of recombinant protein samples was assayed utilizing the Limulus assay (LAL kit; Associates of Cape Cod) according to the manufacturer's instructions. Samples of affinity-purified pMal-c protein and pMA1870-2680 were found to contain high levels of endotoxin [>50,000 EU/mg protein; EU (endotoxin unit)]. This suggested that MBP- or toxin A repeat-containing fusions with the botulinal C fragment should also contain high levels of endotoxin. Accordingly, removal of endotoxin from affinity-purified pMal-c and pMBot protein preparations was attempted as follows.[0598]

Samples of pMal-c and pMBot protein were depyrogenated with polymyxin to determine if the endotoxin could be easily removed. The following amount of protein was treated: 29 ml at 4.8 OD[0599]₂₈₀/ml for pMal-c and 19 mls at 1.44 OD₂₈₀/ml for pMBot. The protein samples were dialyzed extensively against PBS and mixed in a 50 ml tube (Falcon) with 0.5 ml PBS-equilibrated polymyxin B (Affi-Prep Polymyxin, BioRad). The samples were allowed to mix by rotating the tubes overnight at 4° C. The polymyxin was pelleted by centrifugation for 30 min in a bench top centrifuge at maximum speed (approximately 2000×g) and the supernatant was removed. The recovered protein (in the supernatant) was quantified by OD₂₈₀, and the endotoxin activity was assayed by LAL. In both cases only approximately ⅓ of the input protein was recovered and the polymyxin-treated protein retained significant endotoxin contamination (approximately 7000 EU/mg of pMBot).

The depyrogenation experiment was repeated using an independently purified pMal-c protein preparation and similar results were obtained. From these studies it was concluded that significant levels of endotoxin copurifies with these MBP fusion proteins using the amylose resin. Furthermore, this endotoxin cannot be easily removed by polymyxin treatment.[0600]

These results suggest that the presence of the MBP sequences on the fusion protein complicated the removal of endotoxin from preparations of the pMBot protein.[0601]

b) Generation of[0602]C. botulinumC Fragment Protein Free of the MBP

It was demonstrated that the pMBot fusion protein could not be easily purified from contaminating endotoxin in section a) above. The ability to produce a pyrogen-free (e.g., endotoxin-free) preparation of soluble botulinal C fragment protein free of the MBP tag was next investigated. The pMBot expression construct was designed to facilitate purification of the botulinal C fragment from the MBP tag by cleavage of the fusion protein by utilizing an engineered Factor Xa cleavage site present between the MBP and the botulinal C fragment. The Factor Xa cleavage was performed as follows.[0603]

Factor Xa (New England Biolabs) was added to the pMBot protein (using a 0.1-1.0% Factor Xa/pMBot protein ratio) in a variety of buffer conditions [e.g., PBS-NaCl (PBS containing 0.5 M NaCl), PBS-NaCl containing 0.2[0604]% Tween 20, PBS, PBS containing 0.2% Tween 20, PBS-C (PBS containing 2 mM CaCl₂), PBS-C containing either 0.1 or 0.5% Tween 20, PBS-C containing either 0.1 or 0.5% NP-40, PBS-C containing either 0.1 or 0.5% Triton X-100, PBS-C containing 0.1% sodium deoxycholate, PBS-C containing 0.1% SDS]. The Factor Xa digestions were incubated for 12-72 hrs at room temperature.

The extent of cleavage was assessed by Western blot or Coomassie blue staining of proteins following electrophoresis on denaturing SDS-PAGE gels, as described in Example 22. Cleavage reactions (and control samples of uncleaved pMBot protein) were centrifuged for 2 min in a microfuge to remove insoluble protein prior to loading the samples on the gel. The Factor Xa treated samples were compared with uncleaved, uncentrifuged pMBot samples on the same gel. The results of this analysis is summarized below.[0605]

1) Most (about 90%) pMBot protein could be removed by centrifugation, even when uncleaved control samples were utilized. This indicated that the pMBot fusion protein was not fully soluble (i.e., it exists as a suspension rather than as a solution). [This result was consistent with the observation that most affinity-purified pMBot protein precipitates after long term storage (>2 weeks) at 4° C. Additionally, the majority (i.e., 75%) of induced pMBot protein remains in the pellet after sonication and clarification of the induced[0606]E. coli. Resuspension of these insoluble pellets in PBS followed by sonication results in partial solubilization of the insoluble pMBot protein in the pellets.]

2) The portion of pMBot protein that is fully in solution (about 10% of pMBot protein) is completely cleaved by Factor Xa, but the cleaved (released) botulinal C fragment is relatively insoluble such that only the cleaved MBP remains fully in solution.[0607]

3) None of the above reaction conditions enhanced solubility without also reducing effective cleavage. Conditions that effectively solubilized the cleaved botulinal C fragment were not identified.[0608]

4) The use of 0.1% SDS in the buffer used for Factor Xa cleavage enhanced the solubility of the pMBot protein (all of pMBot protein was soluble). However, the presence of the SDS prevented any cleavage of the fusion protein with Factor Xa.[0609]

5) Analysis of pelleted protein from the cleavage reactions indicated that both full length pMBot (i.e., uncleaved) and cleaved botulinal C fragment protein precipitated during incubation.[0610]

These results demonstrate that purification of soluble botulinal C fragment protein after cleavage of the pMBot fusion protein is complicated by the insolubility of both the pMBot protein and the cleaved botulinal C fragment protein.[0611]

c) Expression of[0612]C. botulinumC Fragment Using Various Expression Vectors

In order to determine if the solubility of the botulinal C fragment was enhanced by expressing the C fragment protein as a native protein, an N-terminal His-tagged protein or as a fusion with glutathione-S-transferase (GST), alternative expression plasmids were constructed. These expression constructs were generated utilizing the methodologies described in Example 22. FIG. 27 provides a schematic representation of the vectors described below.[0613]

In FIG. 27, the following abbreviations are used. pP refers to the pET23 vector. pHIS refers to the pETHisa vector. pBlue refers to the pBluescript vector. pM refers to the pMAL-c vector and pG refers to the pGEX3T vector (described in Example 11). The solid black lines represent[0614]C. botulinumC fragment gene sequences; the solid black ovals represent the MBP; the hatched ovals represent GST; “HHHHH” represents the poly-histidine tag. In FIG. 27, when the name for a restriction enzyme appears inside parenthesis, this indicates that the restriction site was destroyed during construction. An asterisk appearing with the name for a restriction enzyme indicates that this restriction site was recreated at a cloning junction.

i) Construction of pPBot[0615]

In order to express the[0616]C. botulinumC fragment as a native (i.e., non-fused) protein, the pPBot plasmid (shown schematically in FIG. 27) was constructed as follows. The C fragment sequences present in pAlterBot (Example 22) were removed by digestion of pAlterBot with NcoI and HindIII. The NcoI/HindIII C fragment insert was ligated to pETHisa vector (described in Example 18b) which was digested with NcoI and HindIII. This ligation creates an expression construct in which the NcoI-encoded methionine of the botulinal C fragment is the initiator codon and directs expression of the native botulinal C fragment. The ligation products were used to transform competent BL21(DE3)pLysS cells (Novagen). Recombinant clones were identified by restriction mapping.

ii) Construction of pHisBot[0617]

In order to express the[0618]C. botulinumC fragment containing a poly-histidine tag at the amino-terminus of the recombinant protein, the pHisBot plasmid (shown schematically in FIG. 27) was constructed as follows. The NcoI/HindIII botulinal C fragment insert from pAlterbot was ligated into the pETHisa vector which was digested with NheI and HindIII. The Ncol (on the C fragment insert) and NheI (on the pETHisa vector) sites were filled in using the Klenow fragment prior to ligation; these sites were then blunt end ligated (the NdeI site was regenerated at the clone junction as predicted). The ligation products were used to transform competent BL21(DE3)pLysS cells and recombinant clones were identified by restriction mapping.

The resulting pHisBot clone expresses the botulinal C fragment protein with a histidine-tagged N-terminal extension having the following sequence: MetGlyH is His HisHisHisHisHisHisHisHisSerSerGlyHislIleGluGlyArgHisMetAla (SEQ ID NO:24); the amino acids encoded by the botulinal C fragment gene are underlined and the vector encoded amino acids are presented in plain type. The nucleotide sequence present in the pETHisa vector which encodes the pHisBot fusion protein is listed in SEQ ID NO:25. The amino acid sequence of the pHisBot protein is listed in SEQ ID NO:26.[0619]

iii) Construction of pGBot[0620]

The botulinal C fragment protein was expressed as a fusion with the glutathione-S-transferase protein by constructing the pGBot plasmid (shown schematically in FIG. 27). This expression construct was created by cloning the NotIl/SalI C fragment insert present in pBlueBot (Example 22) into the pGEX3T vector which was digested with SmaI and XhoI. The NotI site (present on the botulinal fragment) was made blunt prior to ligation using the Klenow fragment. The ligation products were used to transform competent BL21 cells.[0621]

Each of the above expression constructs were tested by restriction digestion to confirm the integrity of the constructs.[0622]

Large scale (1 liter) cultures of pPBot [BL21(DE3)pLysS host], pHisBot [BL21(DE3)pLysS host] and pGBot (BL21 host) were grown in 2×YT medium and induced (using IPTG to 0.8-1.0 mM) for 3 hrs as described in Example 22. Total, soluble and insoluble protein preparations were prepared from 1 ml aliquots of each large scale culture [Williams et al. (1994), supra] and analyzed by SDS-PAGE. No obvious induced band was detectable in the pPBot or pHisBot samples by Coomassie staining, while a prominent insoluble band of the anticipated MW was detected in the pGBot sample. Soluble lysates of the pGBot large scale (resuspended in PBS) or pHisBot large scale [resuspended in[0623]Novagen 1× binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9)] cultures were prepared and used to affinity purify soluble affinity-tagged protein as follows.

The pGBot lysate was affinity purified on a glutathione-agarose resin (Pharmacia) exactly as described in Smith and Corcoran [Current Protocols in Molecular Biology, Supplement 28 (1994), pp. 16.7.1-16.7.7]. The pHisBot protein was purified on the His-Bind resin (Novagen) utilizing the His-bind buffer kit (Novagen) exactly as described by manufacturer.[0624]

Samples from the purification of both the pGBot and pHisBot proteins (including uninduced, induced, total, soluble, and affinity-purified eluted protein) were resolved on SDS-PAGE gels. Following electrophoresis, proteins were analyzed by Coomassie staining or by Western blot detection utilizing a chicken anti-[0625]C. botulinumType A toxoid antibody (as described in Example 22).

These studies showed that the pGBot protein was almost entirely insoluble under the utilized conditions, while the pHisBot protein was soluble. Affinity purification of the pHisBot protein on this first attempt was inefficient, both in terms of yield (most of the immunoreactive botulinal protein did not bind to the His-bind resin) and purity (the botulinal protein was estimated to comprise approximately 20% of the total eluted protein).[0626]

d) Purification of Soluble[0627]C. botulinumC Fragment Protein Substantially Free of Endotoxin Contamination

The above studies showed that the pHisBot protein was expressed in[0628]E. colias a soluble protein. However, the affinity purification of this protein on the His-bind resin was very inefficient. In order to improve the affinity purification of the soluble pHisBot protein (in terms of both yield and purity), an alternative poly-histidine binding affinity resin (Ni-NTA resin; Qiagen) was utilized. The Ni-NTA resin was reported to have a superior binding affinity (Kd=1×10⁻¹³at pH 8.0; Qiagen user manual) relative to the His-bind resin.

A soluble lysate (in[0629]Novagen 1× binding buffer) from an induced 1liter 2× YT culture was prepared as described above. Briefly, the culture of pHisBot [B121(DE3)pLysS host] was grown at 37° C. to an OD₆₀₀of 0.7 in 1 liter of 2×YT medium containing 100 μg/ml ampicillin, 34 μg/ml chloramphenicol and 0.2% glucose. Protein expression was induced by the addition of IPTG to 1 mM. Three hours after the addition of the IPTG, the cells were cooled for 15 min in a ice water bath and then centrifuged 10 min at 5000 rpm in a JA10 rotor (Beckman) at 4° C. The pellets were resuspended in a total volume of 40mls Novagen 1× binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9), transferred to two 35 ml Oakridge tubes and frozen at −70° C. for at least 1 hr. The tubes were thawed and the cells were lysed by sonication (4×20 second bursts using a Branson Sonifier 450 with a power setting of 6-7) on ice. The suspension was clarified by centrifugation for 20 min at 9,000 rpm (10,000×g) in a JA-17 rotor (Beckman).

The soluble lysate was brought to 0.1% NP40 and then was batch absorbed to 7 ml of a 1:1 slurry of Ni-NTA resin:binding buffer by stirring for 1 hr at 4° C. The slurry was poured into a column having an internal diameter of 1 or 2.5 cm (BioRad). The column was then washed sequentially with 15 mls of[0630]Novagen 1× binding buffer containing 0.1% NP40, 15 ml ofNovagen 1× binding buffer, 15 ml wash buffer (60 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9) and 15 ml NaHPO₄wash buffer (50 mM NaHPO₄, pH 7.0, 0.3 M NaCl, 10% glycerol). The bound protein was eluted by protonation of the resin using elution buffer (50 mM NaHPO₄, pH 4.0, 0.3 M NaCl, 10% glycerol). The eluted protein was stored at 4° C.

Samples of total, soluble and eluted protein were resolved by SDS-PAGE. Protein samples were prepared for electrophoresis as described in Example 22b. Duplicate gels were stained with Coomassie blue to visualize the resolved proteins and[0631]C. botulinumtype A toxin-reactive protein was detected by Western blot analysis as described in Example 22b. A representative Coomassie stained gel is shown in FIG. 28. In FIG. 28, the following samples were loaded on the 12.5% acrylamide gel. Lanes 1-4 contain respectively total protein, soluble protein, soluble protein present in the flow-through of the Ni-NTA column and affinity-purified pHisBot protein (i.e., protein released from the Ni-NTA resin by protonation).Lane 5 contains high molecular weight protein markers (BioRad).

The purification of pHisBot protein resulted in a yield of 7 mg of affinity purified protein from a 1 liter starting culture of BL21(DE3)pLysS cells harboring the pHisBot plasmid. The yield of purified pHisBot protein represented approximately 0.4% of the total soluble protein in the induced culture. Analysis of the purified pHisBot protein by SDS-PAGE revealed that at least 90-95% of the protein was present as a single band (FIG. 28) of the predicted MW (50 kD). This 50 kD protein band was immunoreactive with anti-[0632]C. botulinumtype A toxin antibodies. The extinction coefficient of the protein preparation was determined to be 1.4 (using the Pierce BCA assay) or 1.45 (using the Lowry assay) OD₂₈₀per 1 mg/ml solution.

Samples of pH neutralized eluted pHisBot protein were resolved on a KB 803 HPLC column (Shodex). Although His-tagged proteins are retained by this sizing column (perhaps due to the inherent metal binding ability of the proteins), the relative mobility of the pHisBot protein was consistent with that expected for a non-aggregated protein in solution. Most of the induced pHisBot protein was determined to be soluble under the growth and solubilization conditions utilized above (i.e., greater than 90% of the pHisBot protein was found to be soluble as judged by comparison of the levels of pHisBot protein seen in total and soluble protein samples prepared from BL21(DE3)pLysS cells containing the pHisBot plasmid). SDS-PAGE analysis of samples obtained after centrifugation, extended storage at −20° C., and at least 2 cycles of freezing and thawing detected no protein loss (due to precipitation), indicating that the pHisBot protein is soluble in the elution buffer (i.e., 50 mM NaHPO[0633]₄, pH 4.0, 0.3 M NaCl, 10% glycerol).

Determination of endotoxin contamination in the affinity purified pHisBot preparation (after pH neutralization) using the LAL assay (Associates of Cape Cod) detected no significant endotoxin contamination. The assay was performed using the endpoint chromogenic method (without diazo-coupling) according to the manufacturer's instructions. This method can detect concentrations of endotoxin greater than or equal to 0.03 EU/ml (EU refers to endotoxin units). The LAL assay was run using 0.5 ml of a solution comprising 0.5 mg pHisBot protein in 50 mM NaHPO[0634]₄, pH 7.0, 0.3 M NaCl, 10% glycerol; 30-60 EU were detected in the 0.5 ml sample. Therefore, the affinity purified pHisBot preparation contains 60-120 EU/mg of protein. FDA Guidelines for the administration of parenteral drugs require that a composition to be administered to a human contain less than 5 EU/kg body weight (The average human body weight is 70 kg; therefore up to 349 EU units can be delivered in a parental dose.). Because very small amount of protein are administered in a vaccine preparation (generally in the range of 10-500 μg of protein), administration of affinity purified pHisBot containing 60-120 EU/mg protein would result in delivery of only a small percentage of the permissible endotoxin load. For example, administration of 10-500 μg of purified pHisBot to a 70 kg human, where the protein preparation contains 60 EU/mg protein, results in the introduction of only 0.6 to 30 EU [i.e., 0.2 to 8.6% of the maximum allowable endotoxin burden per parenteral dose (less than 5 EU/kg body weight)].

The above results demonstrate that endotoxin (LPS) does not copurify with the pHisBot protein using the above purification scheme. Preparations of recombinantly produced pHisBot protein containing lower levels of endotoxin (less than or equal to 2 EU/mg recombinant protein) may be produced by washing the Ni-NTA column with wash buffer until the OD[0635]₂₈₀returns to baseline levels (i.e., until no more UV-absorbing material comes off of the column).

The above results illustrate a method for the production and purification of soluble, botulinal C fragment protein substantially free of endotoxin.[0636]

Example 25Optimization of the Expression and Purification of pHisBot Protein

The results shown in Example 24d demonstrated that the pHisBot protein is an excellent candidate for use as a vaccine as it could be produced as a soluble protein in[0637]E. coliand could be purified free of pyrogen activity. In order to optimize the expression and purification of the pHisBot protein, a variety of growth and purification conditions were tested.

a) Growth Parameters[0638]

i) Host Strains[0639]

The influence of the host strain utilized upon the production of soluble pHisBot protein was investigated. A large scale purification of pHisBot was performed [as described in Example 24d above] using the BL21(DE3) host (Novagen) rather than the BL21(DE3)pLysS host. The deletion of the pLysS plasmid in the BL21(DE3) host yielded higher levels of expression due to de-repression of the plasmid's T7-lac promoter. However, the yield of affinity-purified soluble recombinant protein was very low (approximately 600 μg/liter culture) when purified under conditions identical to those described in Example 24d above. This result was due to the fact that expression in the BL21(DE3) host yielded very high level expression of the pHisBot protein as insoluble inclusion bodies as shown by SDS-PAGE analysis of protein prepared from induced BL21(DE3) cultures (FIG. 29, lanes 1-7, described below). These results demonstrate that the pHisBot protein is not inherently toxic to[0640]E. colicells and can be expressed to high levels using the appropriate promoter/host combination.

FIG. 29 shows a Coomassie blue stained SDS-PAGE gel (12.5% acrylamide) onto which extracts prepared from BL21(DE3) cells containing the pHisBot plasmid were loaded. Each lane was loaded with 2.5 μl protein sample mixed with 2.5 μl of 2× SDS sample buffer. The samples were handled as described in Example 22b. The following samples were applied to the gel. Lanes 1-7 contain protein isolated from the BL21(DE3) host. Lanes 8-14 contain proteins isolated from the BL21(DE3)pLysS host. Total protein was loaded in[0641]

lanes

1, 2, 4, 6, 8, 10 and 12. Soluble protein was loaded in

Lanes

3, 5, 7, 9, 11 and 13.Lane 1 contains protein from uninduced host cells. Lanes 2-13 contain protein from host cells induced for 3 hours. IPTG was added to a final concentration of 0.1 mM (Lanes 6-7), 0.3 mM (Lanes 4-5) or 1.0 mM (

Lanes

2, 3, 8-13). The cultures were grown in LB broth (Lanes 8-9), 2× YT broth (Lanes 10-11) or terrific broth (Lanes 1-7, 12-13). The pHisBot protein seen in

Lanes

3, 5 and 7 is insoluble protein which spilled over from

Lanes

2, 4 and 6, respectively. High molecular weight protein markers (BioRad) were loaded inLane 14.

A variety of expression conditions were tested to determine if the BL21(DE3) host could be utilized to express soluble pHisBot protein at suitably high levels (i.e., about 10 mg/ml). The conditions altered were temperature (growth at 37 or 30° C.), culture medium (2× YT, LB or Terrific broth) and inducer levels (0.1, 0.3 or 1.0 mM IPTG). All combinations of these variables were tested and the induction levels and solubility was then assessed by SDS-PAGE analysis of total and soluble extracts [prepared from 1 ml samples as described in Williams et al., (1994), supra].[0642]

All cultures were grown in 15 ml tubes (Falcon #2057). All culture medium was prewarmed overnight at the appropriate temperature and were supplemented with 100 μg/ml ampicillin and 0.2% glucose. Terrific broth contains 12 g/l bacto-tryptone, 24 g/l bacto-yeast extract and 100 ml/l of a solution comprising 0.17 M KH[0643]₂PO₄, 0.72 M K₂HPO₄. Cultures were grown in a incubator on a rotating wheel (to ensure aeration) to an OD₆₀₀of approximately 0.4, and induced by the addition of IPTG. In all cases, high level expression of insoluble pHisBot protein was observed, regardless of temperature, medium or inducer concentration.

The effect of varying the concentration of IPTG upon 2× YT cultures grown at 23° C. was then investigated. IPTG was added to a final concentration of either 1 mM, 0.1 mM, 0.05 mM or 0.01 mM. At this temperature, similar levels of pHis Bot protein was induced in the presence of either 1 or 0.1 mM IPTG; these levels of expression was lower than that observed at higher temperatures. Induced protein levels were reduced at 0.05 mM IPTG and absent at 0.01 mM IPTG (relative to 1.0 and 0.1 mM IPTG inductions at 23° C.). However, no conditions were observed in which the induced pHisBot protein was soluble in this host. Thus, although expression levels are superior in the BL21(DE3) host (as compared to the BL21(DE3)pLysS host), conditions that facilitate the production of soluble protein in this host could not be identified.[0644]

These results demonstrate that production of soluble pHisBot protein was achieved using the BL21(DE3)pLysS host in conjunction with the T7-lac promoter.[0645]

ii) Effect of Varying Temperature, Medium and IPTG Concentration and Length of Induction[0646]

The effect growing the host cells in various mediums upon the expression of recombinant botulinal protein from the pHisBot expression construct [in the BL21(DE3)pLysS host] was investigated. BL21(DE3)pLysS cells containing the pHisBot plasmid were grown in either LB, 2× YT or Terrific broth at 37° C. The cells were induced using 1 mM IPTG for a 3 hr induction period. Expression of pHisBot protein was found to be the highest when the cells were grown in 2× YT broth (see FIG. 29, lanes 8-13).[0647]

The cells were then grown at 30° C. in 2× YT broth and the concentration of IPTG was varied from 1.0, 0.3 or 0.1 mM and the length of induction was either 3 or 5 hours. Expression of pHisBot protein was similar at all 3 inducer concentrations utilized and the levels of induced protein were higher after a 5 hr induction as compared to a 3 hr induction.[0648]

Using the conditions found to be optimal for the expression of pHisBot protein, a large scale culture was grown in order to provide sufficient material for a large scale purification of the pHisBot protein. Three 1 liter cultures were grown in 2× YT medium containing 100 μg/ml ampicillin, 34 μg/ml chloramphenicol and 0.2% glucose. The cultures were grown at 30° C. and were induced with 1.0 mM IPTG for a 5 hr period. The cultures were harvested and a soluble lysate were prepared as described in Example 18. A large scale purification was performed as described in Example 24d with the exception that except the soluble lysate was batch absorbed for 3 hours rather than for 1 hour. The final yield was 13 mg pHisBot protein/liter culture. The pHisBot protein represented 0.75% of the total soluble protein.[0649]

The above results demonstrate growth conditions under which soluble pHisBot protein is produced (i.e., use of the BL21(DE3)pLysS host, 2× YT medium, 30° C., 1.0 mM IPTG for 5 hours).[0650]

b) Optimization of Purification Parameters[0651]

For optimization of purification conditions, large scale cultures (3×1 liter) were grown at 30° C. and induced with 1 mM IPTG for 5 hours as described above. The cultures were pooled, distributed to centrifuge bottles, cooled and pelleted as described in Example 24d. The cell pellets were frozen at −70° C. until used. Each cell pellet represented ⅓ of a liter starting culture and individual bottles were utilized for each optimization experiment described below. This standardized the input bacteria used for each experiment, such that the yields of affinity purified pHisBot protein could be compared between different optimization experiments.[0652]

i) Binding Specificity (pH Protonation)[0653]

A lysate of pHisBot culture was prepared in PBS (pH 8.0) and applied to a 3 ml Ni-NTA column equilibrated in PBS (pH 8.0) using a flow rate of 0.2 ml/min (3-4 column volumes/hr) using an Econo chromatography system (BioRad). The column was washed with PBS (pH 8.0) until the absorbance (OD[0654]₂₈₀) of the elute was at baseline levels. The flow rate was then increased to 2 ml/min and the column was equilibrated in PBS (pH 7.0). A pH gradient (pH 7.0 to 4.0 in PBS) was applied in order to elute the bound pHisBot protein from the column. Fractions were collected and aliquots were resolved on SDS-PAGE gels. The PAGE gels were subjected to Western blotting and the pHisBot protein was detected using a chicken anti-C botulinum Type A toxoid antibody as described in Example 22.

From the Western blot analysis it was determined that the pHisBot protein begins to elute from the Ni-NTA column at pH 6.0. This is consistent with the predicted elution of a His-tagged protein monomer at pH 5.9.[0655]

These results demonstrate that the pH at which the pHisBot protein is protonated (released) from Ni-NTA resin in PBS buffer is pH 6.0.[0656]

ii) Binding Specificity (Imidazole Competition)[0657]

In order to define purification conditions under which the native[0658]E. coliproteins could be removed from the Ni-NTA column while leaving the pHisBot protein bound to the column, the following experiment was performed. A lysate of pHisBot culture was prepared in 50 mM NaHPO₄, 0.5 M NaCl, 8 mM imidazole (pH 7.0). This lysate was applied to a 3 ml Ni-NTA column equilibrated in 50 mM NaHPO₄, 0.5 M NaCl (pH 7.0) using an Econo chromatography system (BioRad). A flow rate of 0.2 ml/min (3-4 column volumes/hr) was utilized. The column was washed with 50 mM NaHPO₄, 0.5 M NaCl (pH 7.0) until the absorbance of the elute returned to baseline. The flow rate was then increased to 2 ml/min.

The column was eluted using an imidazole step gradient [in 50 mM NaHPO[0659]₄, 0.5 M NaCl (pH 7.0)]. Elution steps were 20 mM, 40 mM, 60 mM, 80 mM, 100 mM, 200 mM, 1.0 M imidazole, followed by a wash using 0.1 mM EDTA (to strip the nickel from the column and remove any remaining protein). In each step, the wash was continued until the OD₂₈₀returned to baseline. Fractions were resolved on SDS-PAGE gels, Western blotted, and pHisBot protein detected using a chicken anti-C. botulinumType A toxoid antibody as described in Example 22. Duplicate gels were stained with Coomassie blue to detect eluted protein in each fraction.

The results of the PAGE analysis showed that most of the non-specifically binding bacterial protein was removed by the 20 mM imidiazole wash, with the remaining bacterial proteins being removed in the 40 and 60 mM imidazole washes. The pHisBot protein began to elute at 100 mM imidazole and was quantitatively eluted in 200 mM imidazole.[0660]

These results precisely defined the window of imidazole wash stringency that optimally removes[0661]E. coliproteins from the column while specifically retaining the pHisBot protein in this buffer. These results provided conditions under which the pHisBot protein can be purified free of contaminating host proteins.

iii) Purification Buffers and Optimized Purification Protocols[0662]

A variety of purification parameters were tested during the development of an optimized protocol for batch purification of soluble pHisBot protein. The results of these analyses are summarized below.[0663]

Batch purifications were performed (as described in Example 24d) using several buffers to determine if alternative buffers could be utilized for binding of the pHisBot protein to the Ni-NTA column. It was determined that quantitative binding of pHisBot protein to the Ni-NTA resin was achieved in either Tris-HCl (pH 7.9) or NaHPO[0664]₄(pH 8.0) buffers. Binding of the pHisBot protein in NaHPO₄buffer was not inhibited using 5 mM, 8 mM or 60 mM imidazole. Quantitative elution of bound pHisBot protein was obtained in buffers containing 50 mM NaHPO₄, 0.3 M NaCl (pH 3.5-4.0), with or without 10% glycerol. However, quantitation of soluble affinity purified pHisBot protein before and after a freeze thaw (following several weeks storage of the affinity purified elute at −20° C.) revealed that 94% of the protein was recovered using the glycerol-containing buffer, but only 68% of the protein was recovered when the buffer lacking glycerol was employed. This demonstrates that glycerol enhanced the solubility of the pHisBot protein in this low pH buffer when the eluted protein was stored at freezing temperatures (e.g., −20° C.). Neutralization of pH by addition of NaH₂PO₄buffer did not result in obvious protein precipitation.

It was determined that quantitative binding of pHisBot protein using the batch format occurred after 3 hrs (FIG. 30), but not after 1 hr of binding at 4° C. (the resin was stirred during binding). FIG. 30 depicts a Coomaisse blue stained SDS-PAGE gel (7.5% acrylamide) containing samples of proteins isolated during the purification of pHisBot protein from lysate prepared from the BL21(DE3)pLysS host. Each lane was loaded with 5 μl of protein sample mixed with 5 μl of 2× sample buffer and processed as described in Example 22b.[0665]Lane 1 contains high molecular weight protein markers (BioRad).

Lanes

2 and 3 contain protein eluted from the Ni-NTA resin.Lane 4 contains soluble protein after a 3 hr batch incubation with the Ni-NTA resin.

Lanes

5 and 6 contain soluble and total protein, respectively. FIG. 30 demonstrates that the pHisBot protein is completely soluble [compare

Lanes

5 and 6 which show that a similar amount of the 50 kD pHisBot protein is seen in both; if a substantial amount (greater than 20%) of the pHisBot protein were partially insoluble in the host cell, more pHisBot protein would be seen in lane 6 (total protein) as compared to lane 5 (soluble protein)]. FIG. 30 also demonstrates that the pHisBot protein is completely removed from the lysate after batch absorption with the Ni-NTA resin for 3 hours (compareLanes 4 and 5).

The reported high affinity interaction of the Ni-NTA resin with His-tagged proteins (K[0666]_d=1×10⁻ at pH 8.0) suggested that it should be possible to manipulate the resin-protein complexes without significant release of the bound protein. Indeed, it was determined that after the recombinant protein was bound to the Ni-NTA resin, the resin-pHisBot protein complex was highly stable and remained bound following repeated rounds of centrifugation of the resin for 2 min at 1600×g. When this centrifugation step was performed in a 50 ml tube (Falcon), a tight resin pellet formed. This allowed the removal of spent soluble lysate by pouring off the supernatant followed by resuspension of the pellet in wash buffer. Further washes can be performed by centrifugation. The ability to perform additional washes permits the development of protocols for batch absorption of large volumes of lysate with removal of the lysate being performed simply by centrifugation following binding of the recombinant protein to the resin.

A simplified, integrated purification protocol was developed as follows. A soluble lysate was made by resuspending the induced cell pellet in binding buffer [50 mM NaHPO[0667]₄, 0.5 M NaCl, 60 mM imidazole (pH 8.0)], sonicating 4×20 sec and centrifuging for 20 min at 10,000×g. NP-40 was added to 0.1% and Ni-NTA resin (equilibrated in binding buffer) was added. Eight milliliters of a 1:1 slurry (resin:binding buffer) was used per liter of starting culture. The mixture was stirred for 3 hrs at 4° C. The slurry was poured into a column having a 1 cm internal diameter (BioRad), washed with binding buffer containing 0.1% NP40, then binding buffer until baseline was established (these steps may alternatively be performed by centrifugation of the resin, resuspension in binding buffer containing NP40 followed by centrifugation and resuspension in binding buffer). Imidazole was removed by washing the resin with 50 mM NaHPO₄, 0.3M NaCl (pH 7.0). Protein bound to the resin was eluted using the same buffer (50 mM NaHPO₄, 0.3M NaCl) having a reduced pH (pH 3.5-4.0).

A pilot purification was performed following this protocol and yielded 18 mg/liter affinity-purified pHisBot. The pHisBot protein was greater than 90% pure as estimated by Coomassie staining of an SDS-PAGE gel. This represents the highest observed yield of soluble affinity-purified pHisBot protein and this protocol eliminates the need for separate imidazole-containing binding and wash buffers. In addition to providing a simplified and efficient protocol for the affinity purification of recombinant pHisBot protein, the above results provide a variety of purification conditions under which pHisBot protein can be isolated.[0668]

Example 26

The pHisBot Protein is an Effective Immunogen[0669]

In Example 23 it was demonstrated that neutralizing antibodies are generated in mouse serum after nasal immunization with the pMBot protein. However, the pMBot protein was found to copurify with significant amounts of endotoxin which could not be easily removed. The pHisBot protein, in contrast, could be isolated free of significant endotoxin contamination making pHisBot a superior candidate for vaccine production. To further assess the suitability of pHisBot as a vaccine, the immunogenicity of the pHisBot protein was determined and a comparison of the relative immunogenicity of pMBot and pHisBot proteins in mice was performed as follows.[0670]

Two groups of eight BALBc mice were immunized with either pMBot protein or pHisBot protein using Gerbu GMDP adjuvant (CC Biotech). pMBot protein (in PBS containing 10 mM maltose) or pHisBot protein (in 50 mMNaHPO[0671]₄, 0.3 M NaCl, 10% glycerol, pH 4.0) was mixed with Gerbu adjuvant and used to immunize mice. Each mouse received an IP injection of 100 μl antigen/adjuvant mix (50 μg antigen plus 1 μg adjuvant) onday 100 μl. Mice were boosted as described above with the exception that the route of administration was IM on

day

14 and 28. The mice were bled on day 77 and anti-C. botulinumType A toxoid titers were determined using serum collected from individual mice in each group (as described in Example 23). The results are shown in Table 41.

TABLE 41


Anti-C. botulinum Type A Toxoid Serum IgG Titers In Individual
Mice Immunized With pMBot or pHisBot Protein

	Preimmune¹	pMBot²	pHisBot²
	Sample Dilution	Sample Dilution	Sample Dilution

Mouse #	1:50	1:250	1:1250	1:6250	1:50	1:250	1:1250	1:6250	1:50	1:250	1:1250	1:6250

1					0.678	0.190	0.055	0.007	1.574	0.799	0.320	0.093
2					1.161	0.931	0.254	0.075	1.513	0.829	0.409	0.134
3					1.364	0.458	0.195	0.041	1.596	1.028	0.453	0.122
4					1.622	1.189	0.334	0.067	1.552	0.840	0.348	0.090
5					1.612	1.030	0.289	0.067	1.629	1.580	0.895	0.233
6					0.913	0.242	0.069	0.013	1.485	0.952	0.477	0.145
7					0.910	0.235	0.058	0.014	1.524	0.725	0.269	0.069
8					0.747	0.234	0.058	0.014	1.274	0.427	0.116	0.029
Mean	0.048	0.021	0.011	0.002	1.133	0.564	0.164	0.037	1.518	0.896	0.411	0.114
Titer

The results shown above in Table 41 demonstrate that both the pMBot and pHisBot proteins are immunogenic in mice as 100% of the mice (8/8) in each group seroconverted from non-immune to immune status. The results also show that the average titer of anti-[0672]C. botulinumType A toxoid IgG is 2-3 fold higher after immunization with the pHisBot protein relative to immunization with the pMBot protein. This suggests that the pHisBot protein may be a superior immunogen to the pMBot protein.

Example 27Immunization with the Recombinant pHisBot Protein Generates Neutralizing Antibodies

The results shown in Example 26 demonstrated that both the pHisBot and pMBot proteins were capable of inducing high titers of anti-[0673]C. botulinumtype A toxoid-reactive antibodies in immunized hosts. The ability of the immune sera from mice immunized with either the pHisBot or pMBot proteins to neutralizeC. botulinumtype A toxoid in vivo was determined using the mouse neutralization assay described in Example 23b.

The two groups of eight BALBc mice immunized with either pMBot protein or pHisBot protein in Example 26 were boosted again one week after the bleeding on day 77. The boost was performed by mixing pMBot protein (in PBS containing 10 mM maltose) or pHisBot protein (in 50 mM NaHPO[0674]₄, 0.3 M NaCl, 10% glycerol, pH 4.0) with Gerbu adjuvant as described in Example 26. Each mouse received an IP injection of 100 μl antigen/adjuvant mix (50 μg antigen plus 1 μg adjuvant). The mice were bled 6 days after this boost and the serum from mice within a group was pooled. Serum from preimmune mice was also collected (this serum is the same serum described in the footnote to Table 41).

The presence of neutralizing antibodies in the pooled or preimmune serum was detected by challenging mice with 5 LD[0675]₅₀units of type A toxin mixed with 100 μl of pooled serum. The challenge was performed by mixing (per mouse to be injected) 100 μl of serum from each pool with 100 μl of purified type A toxin standard (50 LD₅₀/ml prepared as described in Example 23b) and 500 μl of gel-phosphate. The mixtures were incubated for 30 min at room temperature with occasional mixing. Each of four mice were injected IP with the mixtures (0.7 ml/mouse). The mice were observed for signs of botulism for 72 hours. Mice receiving toxin mixed with serum from mice immunized with either the pHisBot or pMBot proteins showed no signs of botulism intoxication. In contrast, mice receiving preimmune serum died in less than 24 hours.

These results demonstrate that antibodies capable of neutralizing[0676]C. botulinumtype A toxin are induced when either of the recombinantC. botulinumC fragment proteins pHisBot or pMBot are used as immunogens.

Example 28Cloning and Expression of the C Fragment ofC. botulinumSerotype A Toxin inE. coliUtilizing a Native Gene Fragment

In Example 22 above, a synthetic gene was used to express the C fragment of[0677]C. botulinumserotype A toxin inE. coli. The synthetic gene replaced non-preferred (i.e., rare) codons present in the C fragment gene with codons which are preferred byE. coli. The synthetic gene was generated because it was been reported that genes which have a high A/T content (such as most clostridial genes) creates expression difficulties inE. coliand yeast. Furthermore, LaPenotiere et al. suggested that problems encountered with the stability (non-fusion constructs) and solubility (MBP fusion constructs) of the C fragment ofC. botulinumserotype A toxin when expressed inE. coliwas most likely due to the extreme AFr richness of the nativeC. botulinumserotype A toxin gene sequences (LaPenotiere, et al., supra).

In this example, it was demonstrated that successful expression of the C fragment of[0678]C. botulinumtype A toxin gene inE. colidoes not require the elimination of rare codons (i.e., there is no need to use a synthetic gene). This example involved a) the cloning of the native C fragment of theC. botulinumserotype A toxin gene and construction of an expression vector and b) a comparison of the expression and purification yields ofC. botulinumserotype A C fragments derived from native and synthetic expression vectors.

a) Cloning of the Native C Fragment of the[0679]C. botulinumSerotype A Toxin Gene and Construction of an Expression Vector

The serotype A toxin gene was cloned from[0680]C. botulinumgenomic DNA using PCR amplification. The following primer pair was employed: 5′-CGCCATGGCTAG ATTATTATCTACATTTAC-3′ (5′ primer, NcoI site underlined; SEQ ID NO:29) and 5′-GCAAGCTTCTTGACAGACTCATGTAG-3′ (3′ primer, HindIII site underlined; SEQ ID NO:30).C. botulinumtype A strain was obtained from the American Type Culture Collection (ATCC#19397) and grown under anaerobic conditions in Terrific broth medium. High molecular-weightC. botulinumDNA was isolated as described in Example 11. The integrity and yield of genomic DNA was assessed by comparison with a serial dilution of uncut lambda DNA after electrophoresis on an agarose gel.

The gene fragment was cloned by PCR utilizing a proofreading thermostable DNA polymerase (native Pfu polymerase). PCR amplification was performed using the above primer pair in a 50 μl reaction containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl[0681]₂, 200 μM each dNTP, 0.2 μM each primer, and 50 ngC. botulinumgenomic DNA. Reactions were overlaid with 10011 mineral oil, heated to 94° C. 4 min, 0.5 μl native Pfu polymerase (Stratagene) was added, and thirty cycles comprising 94° C. for 1 min, 50° C. for 2 min, 72° C. for 2 min were carried out followed by 10 min at 72° C. An aliquot (10 μl) of the reaction mixture was resolved on an agarose gel and the amplified native C fragment gene was gel purified using the Prep-A-Gene kit (BioRad) and ligated to pCRScript vector DNA (Stratagene). Recombinant clones were isolated and confirmed by restriction digestion, using standard recombinant molecular biology techniques [Sambrook et al. (1989), supra]. In addition, the sequence of approximately 300 bases located at the 5′ end of the C fragment coding region were obtained using standard DNA sequencing methods. The sequence obtained was identical to that of the published sequence.

An expression vector containing the native[0682]C. botulinumserotype A C fragment gene was created by ligation of the NcoI-HindIII fragment containing the C fragment gene from the pCRScript clone to NheI-HindIII restricted pETHisa vector (Example 18b). The NcoI and NheI sites were filled in using the Klenow enzyme prior to ligation; these sites were thus blunt-end ligated together. The resulting construct was termed pHisBotA (native). pHisBotA (native) expresses theC. botulinumserotype A C fragment with a his-tagged N terminal extension which has the following sequence: MetGlyHisHisHisHisHisHisHisHisHisHisSerSerGlyHisIleGluGlyArgHisMetAla (SEQ ID NO:24), where the underlining represents amino acids encoded by theC. botulinumC fragment gene (this N terminal extension contains the recognition site for Factor Xa protease, shown in italics, which can be employed to removed the polyhistdine tract from the N-terminus of the fusion protein). The pHisBot (native) construct expresses the identical protein as the pHisBot construct (Ex. 24c; herein after the pHisBotA) which contains the synthetic gene.

The predicted DNA sequence encoding the native[0683]C. botulinumserotype A C fragment gene contained within pHisBotA (native) is listed in SEQ ID NO:31 [the start of translation (ATG) is located at nucleotides 108-110 and the stop of translation (TAA) is located at nucleotidesl494-1496 in SEQ ID NO:31] and the corresponding amino acid sequence is listed in SEQ ID NO:26 (i.e., the same amino acid sequence as that produced by pHisBotA containing synthetic gene sequences).

b) Comparison of the Expression and Purification Yields of[0684]C. botulinumSerotype A C Fragments Derived from Native and Synthetic Expression Vectors

Recombinant plasmids containing either the native or the synthetic[0685]C. botulinumserotype A C fragment genes were transformed intoE. colistrain B121(DE3) pLysS and protein expression was induced in 1 liter shaker flask cultures. Total protein extracts were isolated, resolved on SDS-PAGE gels andC. botulinumC fragment protein was identified by Western analysis utilizing a chicken anti-C. botulinumserotype A toxoid antiserum as described in Example 22.

Briefly, 1 liter (2XYT+100 μg/ml ampicillin and 34 μg/ml chloramphenicol) cultures of bacteria harboring either the pHisBotA (synthetic) or pHisBotA (native) plasmids in the B121(DE3) pLysS strain were induced to express recombinant protein by addition of IPTG to 1 mM. Cultures were grown at 30-32° C., IPTG was added when the cell density reached an OD[0686]₆₀₀0.5-1.0 and the induced protein was allowed to accumulate for 3-4 hrs after induction.

The cells were cooled for 15 min in a ice water bath and then centrifuged for 10 min at 5000 rpm in a JA10 rotor (Beckman) at 4° C. The cell pellets were resuspended in a total volume of 40[0687]mls 1× binding buffer (40 mM imidazole, 0.5 M NaCl, 50 mM NaPO₄, pH 8.0), transferred to two 50 ml Oakridge tubes and frozen at −70° C. for at least 1 hr. The tubes were then thawed and the cells were lysed by sonication (using four successive 20 second bursts) on ice. The suspension was clarified by centrifugation 20-30 min at 9,000 rpm (10,000 g) in a JA-17 rotor. The soluble lysate was batch absorbed to 7 ml of a 1:1 slurry of NiNTA resin:binding buffer by stirring 2-4 hr at 4° C. The slurry was centrifuged for 1 min at 500 g in 50 ml tube (Falcon), resuspended in 5 mls binding buffer and poured into a 2.5 cm diameter column (BioRad). The column was attached to a UV monitor (ISCO) and the column was washed with binding buffer until a baseline was established. Imidazole was removed by washing with 50 mM NaPO₄, 0.3 M NaCl, 10% glycerol, pH 7.0 and bound protein was eluted using 50 mM NAPO₄, 0.3 M NaCl, 10% glycerol, pH 3.5-4.0.

The eluted proteins were stored at 4° C. Samples of total, soluble, and eluted proteins were resolved by SDS-PAGE. Protein samples were prepared for electrophoresis by mixing 1 μl total (T) or soluble (S) protein with 4 μl PBS and 5[0688]μl 2×SDS-PAGE sample buffer, or 5 μl eluted (E) protein and 5μl 2×SDS-PAGE sample buffer. The samples were heated to 95° C. for 5 min, then cooled and 5 or 10 μls were loaded on 12.5% SDS-PAGE gels. Broad range molecular weight protein markers (BioRad) were also loaded to allow the MW of the identified fusion proteins to be estimated. After electrophoresis, protein was detected either generally by staining gels with Coomassie blue, or specifically, by blotting to nitrocellulose for Western blot detection of specific immunoreactive protein.

For Western blot analysis, the gels were blotted, and protein transfer was confirmed by Ponceau S staining as described in Example 22. After blocking the blots for 1 hr at room temperature in blocking buffer (PBST and 5% milk), 10 ml of a 1/500 dilution of an anti-[0689]C. botulinumtoxin A IgY PEG prep (Ex. 3) in blocking buffer was added and the blots were incubated for an additional hour at room temperature. The blots were washed and developed using a rabbit anti-chicken alkaline phosphatase conjugate (Boehringer Mannheim) as the secondary antibody as described in Ex. 22. This analysis detectedC. botulinumtoxin A-reactive proteins in the pHisBotA (native and synthetic) protein samples (corresponding to the predicted full length proteins identified by Coomassie staining).

A gel containing proteins expressed from the pHisBot and pHisBot (native) constructs during various stages of purification and stained with Coomassie blue is shown in FIG. 31. In FIG. 31, lanes 1-4 and 9 contain proteins expressed by the pHisBotA construct (i.e., the synthetic gene) and lanes 5-8 contain proteins expressed by the pHisBotA (native) construct.[0690]

Lanes

1 and 5 contain total protein extracts;

lanes

2 and 6 contain soluble protein extracts;

lanes

3 and 7 contain proteins which flowed through the NiNTA columns;

lanes

4, 8 and 9 contain protein eluted from the NiNTA columns andlane 10 contains molecular weight markers.

The above purification resulted in a yield of 3 mg (native gene) or 11 mg (synthetic gene) of affinity purified protein from a 1 liter starting culture, of which at least 90-95% of the protein was a single band of the predicted MW (50 kd) and immunoreactivity for recombinant[0691]C. botulinumserotype A C fragment protein. Other than the level of expression, no difference was observed between the native and the synthetic gene expression systems.

These results demonstrate that soluble[0692]C. botulinumserotype A C fragment protein can be expressed inE. coliand purified utilizing either native or synthetic gene sequences.

Example 29Generation of Neutralizing Antibodies Using a RecombinantC. botulinumSerotype A C Fragment Protein Containing a Six Residue His-Tag

In Example 27, neutralizing antibodies were generated utilizing the pHisBotA protein, which contains a histidine-tagged N-terminal extension comprising 10 histidine residues. To determine if the generation of neutralizing antibodies is dependent on the presence of this particular his-tag, a protein containing a shorter N-terminal extension (comprising 6 histidine residues) was produced and tested for the ability to generate neutralizing antibodies. This example involved a) the cloning and expression of the p6HisBotA(syn) protein and b) the generation and characterization of hyperimmune serum.[0693]

a) Cloning and Expression of the p6HisBotA(syn) Protein[0694]

The p6HisBotA(syn) construct was generated as described below; the term “syn” designates the presence of synthetic gene sequences. This construct expresses the C frgament of the[0695]C. botulinumserotype A toxin with a histidine-tagged N terminal extension having the following sequence: MetHisHisHisHisHisHisMetAla (SEQ ID NO:32); the amino acids encoded by the botulinal C fragment gene are underlined and the vector encoded amino acids are presented in plain type. 6× His oligonucleotides [5′-TATGCATCACCATCACCATCA-3′ (SEQ ID NO:33) and 5′-CATGTGATGGTGATGGTGATGCA-3′ (SEQ ID NO:34) were annealed as follows. One microgram of each oligonucleotide was mixed in total of 20μl 1× reaction buffer 2 (NEB) and the mixture was heated at 70° C. for 5 min and then incubated at 42° C. for 5 min. The annealed oligonucleotides were then ligated with gel purified NdeI/HindIII cleaved pET23b (T7 promoter) or pET21b (T71ac promoter) DNA and the gel purified NcoI/HindIIIC. botulinumserotype A C fragment synthetic gene fragment derived from pAlterBot (Ex. 22). Recombinant clones were isolated and confirmed by restriction digestion. The DNA sequence encoding the 6× his-tagged BotA protein contained within p6HisBotA(syn) is listed in SEQ ID NO:35. The amino acid sequence of the p6XHisBotA protein is listed in SEQ ID NO:36.

The resulting recombinant p6XHisBotA plasmid was transformed into the BL21(DE3) pLysS strain, and 1 liter cultures were grown, induced and harvested as described in Example 28. His-tagged protein was purified as described in Example 28, with the following modifications. The binding buffer (BB) contained 5 mM imidazole rather than 40 mM imidazole and NP40 was added to the soluble lysate to a final concentration of 0.1%. The bound material was washed on the column with BB until the baseline was established, then the column was washed successively with BB+20 mM imidazole and BB+40 mM imidazole. The column was eluted as described in Example 28.[0696]

In the case of the pET23-derived expression system, high level expression of insoluble 6HisBotA protein was induced. The pET21-derived vector expressed lower levels of soluble protein that bound the NiNTA resin and eluted in the 40 mM imidazole wash rather than during the low pH elution. These results (i.e., low level expression of a soluble protein) are consistent with the results obtained with pHisBotA protein (Ex. 25); the pHisBotA construct, like the pET21-derived vector, contains the T7lac rather than T7 promoter.[0697]

The 6HisBotA protein thus elutes under less stringent conditions than the IOX histidine-containing pHisBot protein (100-200 mM imidazole; Ex. 25) presumably due to the reduction in the length of the his-tag. The eluted protein was of the predicted size [i.e., slightly reduced in comparison to pHisBotA protein].[0698]

b) Generation and Characterization of Hyperimmune Serum[0699]

Eight BALBc mice were immunized with purified 6HisBotA protein using Gerbu GMDP adjuvant (CC Biotech). The 40 mM imidazole elution was mixed with Gerbu adjuvant and used to immunize mice. Each mouse received a subcutaneous injection of 100 μl antigen/adjuvant mix (12 μg antigen+1 μg adjuvant) on[0700]day 0. Mice were subcutaneously boosted as above onday 14 and bled onday 28. Control mice received pHisBotB protein (prepared as described in Ex. 35 below) in Gerbu adjuvant.

Anti-[0701]C. botulinumserotype A toxoid titers were determined in serum from individual mice from each group using the ELISA described in Example 23a with the exception that the initial testing serum dilution was 1:100 in blocking buffer containing 0.5% Tween 20, followed by serial 5-fold dilutions into this buffer. The results of the ELISA demonstrated that seroconversion (relative to control mice) occurred in all 8 mice.

The ability of the anti-[0702]C. botulinumserotype A C fragment antibodies present in serum from the immunized mice to neutralize nativeC. botulinumtype A toxin was tested using the mouse neutralization assay described in Example 23b. The amount of neutralizing antibodies present in the serum of the immunized mice was determined using serum antibody titrations. The various serum dilutions (0.01 ml) were mixed with 5 LD₅₀units ofC. botulinumtype A toxin and the mixtures were injected IP into mice. The neutralizations were performed in duplicate. The mice were then observed for signs of botulism for 4 days. Undiluted serum was found to protect 100% of the injected mice while the 1:10 diluted serum did not. This corresponds to a neutralization titer of 0.05-0.5 IU/ml.

These results demonstrate that neutralizing antibodies were induced when the 6HisBotA protein was utilized as the immunogen. Furthermore, these results demonstrate that seroconversion and the generation of neutralizing antibodies does not depend on the specific N terminal extension present on the recombinant[0703]C. botulinumtype A C fragment proteins.

Example 30Construction of Vectors for the Expression of His-TaggedC. botulinumType A Toxin C Fragment Protein Using the Synthetic Gene

A number of expression vectors were constructed which contained the synthetic[0704]C. botulinumtype A toxin C fragment gene. These constructs vary as to the promoter (T7 or T7lac) and repressor elements (lacIq) present on the plasmid. The T7 promoter is a stronger promoter than is the T7lac promoter. The various constructs provide varying expression levels and varying levels of plasmid stability. This example involved a) the construction of expression vectors containing the syntheticC. botulinumtype A C fragment gene and b) the determination of the expression level achieved using plasmids containing either the kanamycin resistance or the ampicillin resistance genes in small scale cultures.

a) Construction of Expression Vectors Containing the Synthetic[0705]C. botulinumType A C Fragment Gene

Expression vectors containing the synthetic[0706]C. botulinumtype A C fragment gene were engineered to utilize the kanamycin resistance rather than the ampicillin resistance gene. This was done for several reasons including concerns regarding the presence of residual ampicillin in recombinant protein derived from plasmids containing the ampicillin resistance gene. In addition, ampicillin resistant plasmids are more difficult to maintain in culture; the β-lactamase secreted by cells containing ampicillin resistant plasmids rapidly degrades extracellular ampicillin, allowing the growth of plasmid-negative cells.

A second altered feature of the expression vectors is the inclusion of lacIq gene in the plasmid. This repressor lowers expression from lac regulated promoters (the chromosomally located, lactose regulated T7 polymerase gene and the plasmid located T7lac promoter). This down regulates uninduced protein expression and can enhance the stability of recombinant cell lines. The final alteration to the vectors is the inclusion of either the T7 or 17lac promoters that drive high or moderate level expression of recombinant protein, respectively.[0707]

The expression plasmids were constructed as follows. In all cases, the protein expressed is the pHisBotA(syn) protein previously described, and the only differences between constructs is the alteration of the various regulatory elements described above.[0708]

i) Construction of pHisBotA(syn) kan T7lac[0709]

The pHisBotA(syn) kan T7lac construct was made by inserting the SapI/XhoI fragment containing the[0710]C. botulinumtype A C fragment from pHisBotA(syn) into pET24 digested with SapI/XhoI (Novagen; fragment contains kan gene and origin of replication). The desired construct was selected for kanamycin resistance and confirmed by restriction digestion.

ii) Construction of pHisBotA(syn) kan lacIq T7lac[0711]

The pHisBotA(syn) kan lacIq T7lac construct was made by inserting the XbaI/HindIII fragment containing the[0712]C. botulinumtype A C fragment from pHisBotA(syn)kanT7lac into the pET24a vector digested with XbaI/HindIII. The resulting construct was confirmed by restriction digestion.

iii) Construction of pHisBotA(syn) kan lacIq T7[0713]

The pHisBotA(syn) kan lacIq T7 construct was made by inserting the XbaI/HindIII fragment containing the[0714]C. botulinumtype A C fragment from pHisBotA(syn) kan lacIq T7lac into XbaI/HindIII-digested pHisBotB(syn) kan lacIq T7 (described in Ex 37c below). The resulting construct was confirmed by restriction digestion.

b) Determination of the Expression Level Achieved Using Plasmids Containing Either the Kanamycin Resistance or the Ampicillin Resistance Genes in Small Scale Cultures[0715]

One liter cultures of pHisBotA(syn) kan T7lac/B121(DE3)pLysS and pHisBotA(syn) amp T7lac/B121(DE3)pLysS [this is the previously designated pHisBotA(syn) construct] were grown, induced and his-tagged proteins were purified as described in Example 28. No differences in yield or protein integrity/purity were observed.[0716]

These results demonstrate that the antigen induction levels from expression constructs were not affected by the choice of ampicillin versus kanamycin antibiotic resistance genes.[0717]

Example 31Fermentation of Cells Expressing Recombinant Botulinal Proteins

a) Fermentation Culture of Cells Expressing Recombinant Botulinal Proteins[0718]

Fermentation cultures were grown under the following conditions which were optimized for growth of the BL21(DE3) strains containing pET derived expression vectors. An overnight 1 liter feeder culture was prepared by inoculating of 1 liter media (in a 2 L shaker flask) with a fresh colony grown on an LB kan plate. The feeder culture contained: 600 mls nitrogen source [20 gm yeast extract (BBL) and 40 gm tryptone (BBL)/600 mis], 200[0719]mis 5×fermentation salts (per liter: 48.5 gm K₂HPO₄, 12 gm NaH₂PO₄.H₂O, 5 gm NH₄Cl, 2.5 gm NaCl), 180 mls dH₂O, 20mls 20% glucose, 2 mls 1 M MgSO₄, 5 mls 0.05 μM CaCl, and 4 mls of a 10 mg/ml kanamycin stock. All solutions were sterilized by autoclaving, except the kanamycin stock which was filter sterilized.

An aliquot (5 ml) of the feeder culture broth was removed prior to inoculation, and grown for 2 days at 37° C. as a culture broth sterility control. Growth was not observed in this control culture in any of the fermentations performed.[0720]

The inoculated feeder culture was grown for 12-15 hrs (ON) at 30-37° C. Care was taken to prevent oversaturation of this culture. The saturated feeder culture was added to 10 L of fermentation media in fermenter (BiofloIV, New Brunswick Scientific, Edison, N.J.) as follows. The fermenter was sterilized 120 min at 121° C. with dH[0721]₂O. The sterile water was removed, and fermentation media added as follows: 6 liters nitrogen source, 2liters 5× fermentation salts, 2liters 2% glucose, 20 mls 1 M MgSO₄, 50 mls 0.05 M CaCl₂, 2.5-3.5 mls Macol P 400 antifoam (PPG Industries Inc., Gurnee, Ill.), 40mls 10 mg/ml kanamycin and 10 mls trace elements (8 gm FeSO₄.7H₂O, 2 gm MnSO₄.H₂O, 2 gm AlCl₃.6H₂O, 0.8 gm CoCl.6H,O, 0.4 gm ZnSO₄.7H₂O, 0.4 gm Na₂MoO₄.2H₂O, 0.2 gm CuCl₂.2H₂O, 0.2 gm NiCl₂, 0.1 gm H₃BO₄/200 mls 5 M HCl). All solutions were sterilized by autoclaving, except the kanamycin stock which was filter sterilized. Fermentation media was prewarmed to 37° C. before the addition of the feeder culture.

After the addition of the feeder culture, the culture was fermented at 37° C., 400 rpm agitation, and 10 l/min air sparging. The DO[0722]₂control was set to 20% PID and dissolved oxygen levels were controlled by increasing the rate of agitation from 400-850 rpm under DO₂control. DO₂levels were maintained at greater than or equal to 20% throughout the entire fermentation. When agitation levels reached 500-600 rpm the temperature was lowered to 30° C. to reduce the oxygen consumption rate. Culture growth was continued until endogenous carbon sources were depleted. In these fermentations, glucose was depleted first [monitored with a glucose monitoring kit (Sigma)], followed by assimilation of acetate and other acidic carbons [monitored using an acetate test kit (Boehringer Mannheim)]. During the assimilation phase, the pH rose from 6.6-6.8 (starting pH) to 7.4-7.5, at which time the bulk of the remaining carbon source was depleted. This was signaled by a drop in agitation rate (from a maximum of 700-800 rpm) and a rise in DO₂levels >30%. This corresponds to a OD₆₀₀reading of 18-20/ml. At this point a fed batch mode was initiated, in which a feed solution of 50% glucose was added at a rate of approximately 4 gm glucose/liter/hr. The pH was adjusted to 7.0 by the addition of 25% H₃PO₄(approximately 60 mls). Culture growth was continued and reached peak oxygen consumption within the next 3 hrs of growth (while the remaining residual non-glucose carbon sources were assimilated). This phase is characterized by a slow increase in pH, and air sparging was increased to 15 L/min, to keep the maximum rpm below 850.

Once the residual acidic carbon sources are depleted the agitation rate decreases to 650-750 rpm and the pH begins to drop. pH control was maintained at 7.0 PID by regulated pump addition of a sterile 4M NaOH solution which was consumed at a steady rate for the remainder of the fermentation. Growth was continued at 30° C., and the cultures were grown linearly at a growth rate of 4-7 OD[0723]₆₀₀units/hr, to at least 81.5 OD₆₀₀units/ml (>30 g/l dry cell weight) without induction. Antifoam (a 1:1 dilution with filter sterilized 100% ethanol) was added as necessary throughout the fermentation to prevent foaming.

During the fed batch mode, glucose was assimilated immediately (concentration in media consistently less than 0.1 gm/liter) and acetate was not produced in significant levels by the pET plasmid/BL21(DE3) cell lines tested (approximately 1 gm/liter at end of fermentation; this is lower than that observed in harvests from shaker flask cultures utilizing the same strains). This was fortuitous, since high levels of acetate has been shown to inhibit induction levels in a variety of expression systems. The above described conditions were found to be highly reproducible between fermentations and utilizing different expression plasmids. As a result, glucose and acetate level monitoring were no longer preformed during fermentation.[0724]

b) Induction of Fermentation Cultures[0725]

Induction with IPTG (250 mg-10 gms, depending on the expression vector and experiment) was initiated 1-3 hrs after initiation of the glucose feed (30-50 OD[0726]₆₀₀ml). The growth rate after induction was monitored on a hourly basis. Aliquots (5-10 ml) of cells were harvested at the time of induction, and at hourly intervals post-induction. Optical density readings were determined by measuring the absorbance at 600 nm of 10 μl culture in 990 μl PBS versus a PBS control. The growth rate after induction was found to vary depending on the expression system utilized.

c) Monitoring of Fermentation Cultures[0727]

Fermentation cultures were monitored using the following control assays.[0728]

i) Colony Forming Ability[0729]

An aliquots of cells were removed from the cultures at each timepoint sampled (uninduced and at various times after induction) were serially diluted in PBS ([0730]dilution 1=15 μl cells/3 ml PBS,dilution 2=15 μl ofdilution 1/3 ml PBS,dilution 3=3 or 6 μl ofdilution 2/3 mls PBS) and 100 μl ofdilution 3 was plated on an LB or TSA (trypticase soy agar) plate. The plates were incubated ON at 37° C. and then the colonies are counted and scored for macro or micro growth.

ii) Phenotypic Characterization[0731]

Colonies growing on LB or TSA plates (above) from uninduced and induced timepoints were replica plated onto LB+kan, LB+chloramphenicol (for fermentations utilizing LysS or pACYCGro plasmids), LB+kan+1 mM IPTG and LB plates, in this order. The plates were grown 6-8 hrs at 37° C. and growth was scored on each plate for a minimum of 40-50 well isolated colonies. The percentage of cells retaining the plasmid at time of induction (i.e., uninduced cultures immediately prior to the addition of IPTG) was determined to be the # colonies LB+Kan (or chloramphenicol) plate/# colonies[0732]LB plate X 100%. The percentage of cells with mutated pET plasmids was determined to be the # colonies LB+Kan+[PTG plate/# coloniesLB plate X 100%. Colonies on all LB plates were scored morphologically forE. coliphenotype as a contamination control. Morphologically detectable contaminant colonies were not detected in any fermentation.

iii) Recombinant BotA Protein Induction[0733]

A total of 10 OD[0734]₆₀₀units of cells (e.g., 200 μl of cells at OD₆₀₀=50/ml) were removed from each timepoint sample to a 1.5 ml microfuge tube and pelleted for 2 min at maximum rpm in a microfuge. The pellets were resuspended in 1 ml of 50 mM NaHPO₄, 0.5 M NaCl, 40 mM imidazole buffer (pH 6.8) containing 1 mg/ml lysozyme. The samples were incubated for 20 min at room temperature and stored ON at −70° C. Samples were thawed completely at room temperature and sonicated 2×10 seconds with a Branson Sonifier 450 microtip probe at # 3 power setting. The samples were centrifuged for 5 min at maximum rpm in a microfuge.

An aliquot (20 μl) of the protein samples were removed to 20[0735]μl 2× sample buffer, before or after centrifugation, for total and soluble protein extracts, respectively. The samples were heated to 95° C. for 5 min, then cooled and 5 or 10 μl were loaded onto 12.5% SDS-PAGE gels. High molecular weight protein markers (BioRad) were also loaded to allow for estimation of the MW of identified fusion proteins. After electrophoresis, protein was detected either generally by staining gels with Coomassie blue, or specifically, by blotting onto nitrocellulose (as described in Ex. 28) for Western blot detection of specific his-tagged proteins utilizing a NiNTA-alkaline phosphatase conjugate exactly as described by the manufacturer (Qiagen).

iv) Recombinant Antigen Purification[0736]

At the end of each fermentation run, 1-10 liters of culture were harvested from the fermenter and the bacterial cells were pelleted by centrifugation at 6000 rpm for 10 min in a JA10 rotor (Beckman). The cell pellets were stored frozen at −70° C. or utilized immediately without freezing. Cell pellets were resuspended to 15-20% weight to volume in resuspension buffer (generally 50 mM NaPO[0737]₄, 0.5 M NaCl, 40 mM imidazole, pH 6.8) and lysed utilizing either sonication or high pressure homogenization.

For sonication, the resuspension buffer was supplemented with lysozyme to 1 mg/ml, and the suspension was incubated for 20 min. at room temp. The sample was then frozen ON at −70° C., thawed and sonicated 4×20 seconds at microtip maximum to reduce viscosity. For homogenization, the cells were lyzed by 2 passes through a homogenizer (Rannie Mini-lab type 8.30H) at 600 Bar. Cell lysates were clarified by centrifugation for 30 min at 10,000 rpm in a JA10 rotor.[0738]

For IDA chromatography, samples were flocculated utilizing polyethyleneimine (PEI) prior to centrifugation. Cell pellets were resuspended in cell resuspension buffer (CRB: 50 mM NaPO[0739]₄, 0.5 M NaCl, 40 mM imidazole, pH 6.8) to create a 20% cell suspension (wet weight of cells/volume of CRB) and cell lysates were prepared as described above (sonication or homogenization). PEI (a 2% solution in dH₂O, pH 7.5 with HCl) was added to the cell lysate a final concentration of 0.2%, and stirred for 20 min at room temperature prior to centrifugation (8,500 rpm in JA10 rotor for 30 minutes at 4° C.). This treatment removed RNA, DNA and cell wall components, resulting in a clarified, low viscosity lysate (“PEI clarified lysate”).

His-tagged proteins were purified from soluble lysates by metal-chelate affinity chromatography using either a NiNTA resin (as described in Ex. 28) or an IDA (iminodiacetic acid) resin as described below.[0740]

IDA resin affinity purifications were performed utilizing a low pressure chromatography system (ISCO). A 7 ml (small scale) or 70 ml (large scale) Chelating Sepharose Fast Flow (Pharmacia) affinity column was poured; in addition, a second guard column was poured and attached in line with the first column (to capture Ni ions that leached off the affinity column). The columns were washed with 3 column volumes of dH[0741]₂O. The guard column was then removed and the affinity column was washed with 0.3 M NiSO₄until resistivity was established, then with dH₂O until the resistivity returned to baseline. The columns were reconnected and equilibrated with cell resuspension buffer (CRB; 50 mM NaPO₄, 0.5 M NaCl, 40 mM imidazole, pH 6.8). The clarified sample (in CRB) was loaded. Flow rates were 5 ml/min for small scale columns and 20 ml/min for large scale columns. After sample loading, the column was washed with CRB until a baseline established and bound protein was eluted with elution buffer (50 mM NaPO₄, 0.5 M NaCl, 800 mM imidazole, 20% glycerol, pH 6.8 or 8.0). Protein samples were stored at 4° C. or −20° C. The yield of eluted protein was established by measuring the OD₂₈₀of the elutions, with a 1 mg/ml solution of protein assumed to yield an absorbance reading of 2.0.

The IDA columns may be regenerated and reused multiple times (>10). To regenerate the column, the column was washed with 2-3 column volumes of H[0742]₂O, then 0.05 M EDTA until all of the blue/green color was removed followed by a wash with dH₂O. The IDA columns were sterilized with 0.1 M NaOH (using at least 3 column volumes but not more than 50 minutes contact time with column packing material), then washed with 3 column volumes 0.05 M NaPO₄, pH 5.0, then dH₂O and stored at room temperature in 20% ethanol.

Example 32Construction of a Folding Chaperone Overexpression System

Co-overexpression of the[0743]E. coliGroEL/GroES folding chaperones in a cell expressing a recombinant foreign protein has been reported to enhance the solubility of some foreign proteins that are otherwise insoluble when expressed inE. coli(Gragerouu et al. (1992) Proc. Natl. Acad. Sci. USA 89:10344]. The improvement in solubility is thought to be due to chaperone-mediated binding and unfolding of insoluble denatured proteins, thus allowing multiple attempts for productive refolding of recombinant proteins. By overexpressing the chaperones, the unfolding/refolding reaction is driven by excess chaperone, resulting, in some cases, in higher yields of soluble protein.

In this example, a chaperone overexpression system, compatible with pET vector expression systems, was constructed to facilitate testing chaperone-mediated solubilization of[0744]C. botulinumtype A proteins. This example involved the cloning of the GroEL/ES operon and construction of a pLysS-based chaperone hyperexpression system.

The GroEL/GroES operon was PCR amplified and cloned into the pCRScript vector as described in Example 28. The following primer pair was used: 5′-CGCAT ATGAATATTCGTCCATTGCATG-3′ (SEQ ID NO:37) [5′ primer, start codon of groES gene converted to NdeI site (underlined)] and 5′-GGAAGCTTGCAGGGCAAT TACATCATG (SEQ ID NO:38) (3′ primer, stop codon of groEL gene italicized, engineered HindIII site underlined). Following amplification, the chaperone operon was excised as an NdeI/HindIII fragment and cloned into pET23b digested with NdeI and HindIII. This construction places the Gro operon under the control of the T7 promoter of the pET23 vector. The desired construct was confirmed by restriction digestion.[0745]

The T7 promoter-Gro operon-T7 terminator expression cassette was then excised as a BglII/BspEI (filled) fragment and cloned into BamHI (compatible with BglII/HindIII (filled) cleaved pLysS plasmid (this removed the T7 lysozyme gene). The resulting construct was designated pACYCGro, since the plasmid utilizing the pACYC184 origin from the plysS plasmid. Proper construction was confirmed by restriction digestion.[0746]

pACYCGro was transformed into BL21(DE3), cultures were grown and induced with 1 mM IPTG as described in preceding examples. Total and soluble protein extracts were generated from cells removed before and after IPTG induction and were resolved on a 12.5% SDS-PAGE gel and stained with Coomassie blue. This analysis revealed that high levels of soluble GroEl and GroES proteins were made in the induced cells. These results demonstrated that the chaperone hyper-expression system was functional.[0747]

Example 33Growth of BotA/pACYCGro Cell Lines in Fermentation Cultures

Induction of BL21(DE3) cells lacking the LysS plasmid which contained BotA expression constructs grown in shaker flask or fermentation culture resulted in the expression of primarily insoluble BotA protein. Fermentation cultures were performed to determine if the simultaneous overexpression of the Gro operon and recombinant C botulinum type A proteins (BotA proteins) resulted in enhanced solubility of the recombinant BotA protein. This example involved the fermentation of pHisBotA(syn)kan lacIq T7lac/pACYCGro BL21(DE3) and pHisBotA(syn)kan lacIq T7/pACYCGro BL21(DE3) cell lines. The fermentations were repeated exactly as described in Example 31. Chloramphenicol (34 μg/ml) was included in the feeder and fermentation cultures.[0748]

a) Fermentation of pHisBotA(syn)kan lacIq T7lac/pACYCGro BL21(DE3) Cells[0749]

For fermentation of cells containing plasmids comprising the T7lac promoter, induction was with 2 gms IPTG. at 1 hr post initiation of glucose feed. The OD[0750]₆₀₀was 35 at time of induction, then 48.5, 61.5, 67 at 1-3 hrs post induction. Viable colony counts decreased from 0-3 hr induction [21 (13), 0, 0, 0;dilution 3 utilized 3 μl ofdilution 2 cells] with numbers in parenthesis for the indicating microcolonies. Of 28 colonies scored at the time of induction, 23 retained the pHisBotA(syn)kan lacIq T7lac plasmid (kan resistant), 22 contained the chaperone plasmid (chloramphenicol resistant) and no colonies at induction grew on IPTG+Kan plates (no mutations detected). These results were indicative of very strong promoter induction, since colony viability dropped immediately after induction.

Total and soluble extracts were resolved on a 12.5% SDS-PAGE gel and stained with Coomassie. High level induction of Gro chaperones was observed, but very low level expression of soluble BotA protein was observed, increasing from 1 to 4.0 hrs post induction (no expression detected in uninduced cells). The dramatically lower expression of the BotA antigen in the presence of chaperone may be due to promoter occlusion (i.e., the stronger T7 promoter on the chaperone plasmid is preferentially utilized).[0751]

b) Fermentation of pHisBotA(syn)kan lacIq T7/pACYCGro BL21(DE3) Cells[0752]

A fermentation utilizing the T7-driven BotA expression plasmid was performed. Induction was with 1 gm IPTG at 2 hrs post initiation of glucose feed. The OD[0753]₆₀₀was 41 at time of induction, then 51.5, 61.5, 61.5 and 66 at 1-4 hrs post induction. Viable colony counts decreased from 0-4 hrs induction [71, 1 (34), 1 (1), 1, 0;dilution 3 utilized 6μl dilution 2 cells) with numbers in parenthesis for the uninduced timepoint indicating microcolonies. Of 65 colonies scored at the time of induction, all 65 retained both the pHisBotA(syn)kan lacIq T7 plasmid (kan resistant) and the chaperone plasmid (chloramphenicol resistant) and no colonies at induction grew on IPTG+Kan plates (no mutations detected).

Total and soluble extracts were resolved on a 12.5% SDS-PAGE gel and stained with Coomassie. High level induction of Gro chaperones and moderate level expression of soluble BotA protein was observed, increasing from 1 to 4.0 hrs post induction (no expression detected in uninduced cells).[0754]

A PEI-clarified lysate (0.2% final cocnentration PEI) [850 ml from 130 gm cell pellet (2 liters fermentation harvest)] was purified on a large scale IDA column. A total of 78 mg of protein was eluted. Extracts from the purification were resolved on a 12.5% SDS-PAGE gel and stained with Coomassie. The elution was found to contain an approximately 1:1 mix of BotA/chaperone protein (FIG. 32). PEI lysates prepared in this manner were typically 16 OD[0755]₂₈₀/ml. This was estimated to be 8 mg protein/ml of lysate (by BCA assay). Thus, the eluted recombinant BotA protein represented 0.55% of the total soluble cellular protein applied to the column.

In FIG. 32,[0756]lane 1 contains molecular weight markers, lanes 2-9 contain extracts from pHisBotA(syn)kan lacIq L7/pACYCGro/BL21(DE3) cells before or during purification on the IDA column.Lane 2 contains total protein extract;lane 3 contains soluble protein extract;

lanes

4 and 5 contain PEI-clarified lysates (duplicates);

lanes

6 and 7 contain flow-through from the IDA column (duplicates) and

lanes

8 and 9 contain IDA column elute (lane 9 contains {fraction (1/10)} the amount applied to lane 8).

These results demonstrate, that although the majority of the BotA protein produced was insoluble, 20 mg/liter of soluble recombinant BotA protein can be purified utilizing the pHisBotA(syn)kan lacIq T7/pACYCGro/BL21(DE3) expression system.[0757]

Example 34Purification of Recombinant BotA Protein from Folding Chaperones

In this example of size exclusion chromatography was used to purify the recombinant BotA protein away from the folding chaperones and imidazole present in the IDA-purified material (Ex. 33).[0758]

To enhance the solubility of the recombinant BotA protein during scale-up, the protein was co-expressed with folding chaperones (Ex. 33). As observed with the recombinant BotB protein (Example 40 below), the folding chaperones co-eluted with the recombinant BotA protein during the Ni-IDA purification step. Because the recombinant BotA and BotB proteins have similar molecular weights (about {fraction (1/10)} the size of the non-reduced folding chaperone) and the imidazole step gradient strategy was unsuccessful in purifying BotB away from the folding chaperone (see Ex. 40), size exclusion chromatography was examined for the ability to purify the recombinant BotA protein away from the folding chaperones.[0759]

A column (2.5×24 cm) containing Sephacryl S-100 HR (Pharmacia) was poured (bed volume˜110 ml). Proteins having molecular weights greater than 100 K are expected to elute in the void volume under these conditions and smaller proteins should be retained by the beads and elute at different times, depending on their molecular weights. To maintain solubility of the purified BotA protein, the Sephacryl column was equilibrated in a buffer having the same salt concentration as the buffer used to elute the BotA protein from the IDA column (i.e., 50 mM sodium phosphate, 0.5 M NaCl, 10% glycerol; all reagents from Mallinkrodt, Chesterfield, Mo.).[0760]

Five milliliters of the IDA-purified recombinant BotA protein (Ex. 33) was filtered through a 0.45μ syringe filter, applied to the column and the equilibration buffer was pumped through the column at a flow rate of 1 ml/minute. Eluted proteins were monitored by absorbance at 280 nm and collected either manually or with a fraction collector (BioRad). Appropriate fractions were pooled, if necessary, and the protein was quantitated by absorbance at 280 nm and/or BCA protein assay (Pierce). The isolated peaks were then analyzed by native and/or SDS-PAGE to identify the proteins present and to evaluate purity. The folding chaperone eluted first, followed by the recombinant BotA protein and then the imidazole peak.[0761]

SDS-PAGE analysis (12.5% polyacrylamide, reduced samples) was used to evaluate the purity of the IDA-purified recombinant BotA protein before and after S-100 purification. FIG. 33 shows the difference in purity before and after the S-100 purification step. In FIG. 33,[0762]lane 1 contains molecular weight markers (BioRad broad range).Lane 2 shows the IDA-purified recombinant BotA protein preparation, which is contaminated with significant amounts of the folding chaperone. Following S-100 purification, the amount of folding chaperone present in the BotA sample is reduced dramatically (lane 3).Lane 4 contains no protein (i.e., it is a blank lane); lanes 5-8 contain samples of IDA-purified recombinant BotB and BotE proteins and are discussed infra.

Endotoxin levels in the S-100 purified BotA preparation were determined using the LAL assay (Associates of Cape Cod) as describe in Example 24. The purified BotA preparation was found to contain 22.7 to 45.5 EU/mg recombinant protein.[0763]

These results demonstrate that size exclusion chromatography was successful in purifying the recombinant BotA protein from folding chaperones and imidazole following an initial IDA purification step. Furthermore, these results demonstrate that the S-100 purified BotA protein was substantially free of endotoxin.[0764]

Example 35Cloning and Expression of the C Fragment of theC. botulinumSerotype B Toxin Gene

The[0765]C. botulinumtype B neurotoxin gene has been cloned and sequenced [Whelan et al. (1992) Appl. Environ. Microbiol. 58:2345 and Hutson et al. (1994) Curr. Microbiol. 28:101]. The nucleotide sequence of the toxin gene derived from the Eklund 17B strain (ATCC 25765) is available from the EMBL/GenBank sequence data banks under the accession number X71343; the nucleotide sequence of the coding region is listed in SEQ ID NO:39. The amino acid sequence of theC. botulinumtype B neurotoxin derived from the strain Eklund 17B is listed in SEQ ID NO:40. The nucleotide sequence of theC. botulinumserotype B toxin gene derived from the Danish strain is listed in SEQ ID NO:41 and the corresponding amino acid sequence is listed in SEQ ID NO:42.

The DNA sequence encoding the native[0766]C. botulinumserotype B C fragment gene derived from the Eklund 17B strain can be expressed using the pETHisb vector; the resulting coding region is listed in SEQ ID NO:43 and the corresponding amino acid sequence is listed in SEQ ID NO:44. The DNA sequence encoding the native C botulinum serotype B C fragment gene derived from the Danish strain can be expressed using the pETHisb vector; the resulting coding region is listed in SEQ ID NO:45 and the corresponding amino acid sequence is listed in SEQ ID NO:46. The C frgarnent region from any strain ofC. botulinumserotype B can be amplified and expressed using the approach illustrated below using the C fragment derived fromC. botulinumtype B 2017 strain.

The[0767]C. botulinumtype B neurotoxin gene is synthesized as a single polypeptide chain which is processed to form a dimer composed of a light and a heavy chain linked via disulfide bonds; the type B neurotoxin has been reported to exist as a mixture of predominatly single chain with some double chain (Whelan et al., supra). The 50 kD carboxy-terminal portion of the heavy chain is referred to as the C fragment or the H_Cdomain. Expression of the C fragment ofC. botulinumtype B toxin in heterologous hosts (e.g.,E. coli) has not been previously reported.

The native C fragment of the[0768]C. botulinumserotype B toxin gene was cloned and expression constructs were made to facilitate protein expression inE. coli. This example involved PCR amplification of the gene, cloning, and construction of expression vectors.

The C fragment of the[0769]C. botulinumserotype B (BotB) toxin gene was cloned using the protocols and conditions described in Example 28 for the isolation of the native BotA gene. TheC. botulinumtype B 2017 strain was obtained from the American Type Culture Collection (ATCC #17843). The following primer pair was used to amplify the BotB gene: 5′-CGCCATGGCTGATACAATACTAATAGAA ATG-3′ [5′ primer, engineered NcoI site underlined (SEQ ID NO:47)] and 5′-GCAAG CTT7TATTCAGTCCACCCTTCATC-3′ [3′ primer, engineered HindIII site underlined, native gene termination codon italicized (SEQ ID NO:48)]. After cloning into the pCRscript vector, the Nhe[(filled)/HindIII fragment was cloned into pETHisb vector as described for BotA C fragment gene in Example 28. The resulting construct was termed pHisBotB.

pHisBotB expresses the BotB gene sequences under the transcriptional control of the T7 lac promoter and the resulting protein contains an N-terminal 10× His-tag affinity tag. The pHisBotB expression construct was transformed into BL21(DE3) pLysS competent cells and 1 liter cultures were grown, induced and his-tagged proteins were purified utilizing a NiNTA resin (eluted in low pH elution buffer) as described in Example 28. Total, soluble and purified proteins were resolved by SDS-PAGE and detected by Coomassie staining and Western blot hybridization utilizing a chicken anti-[0770]C. botulinumserotype B toxoid primary antibody (generated by immunization of hens usingC. botulinumserotype B toxoid as described in Example 3). Samples of BotA and BotE C fragment proteins were included on the gels for MW and immunogenicity comparisons. Strong immunoreactivity to only the BotB protein was detected with the anti-C. botulinumserotype B toxoid antibodies. The recombinant BotB protein was expressed at low levels (3 mg/liter) as a soluble protein. The purified BotB protein migrated as a single band of the predicted MW (i.e., −50 kD).

These results demonstrate the cloning of the native[0771]C. botulinumserotype B C fragment gene, the expression and purification of the recombinant BotB protein as a soluble his-tagged protein inE. coli.

Example 36Generation of Neutralizing Antibodies Using the Recombinant pHisBotB Protein

The ability of the purified pHisBot protein to generate neutralizing antibodies was examined. Nine BALBc mice were immunized with BotB protein (purified as described in Ex. 35) using Gerbu GMDP adjuvant (CC Biotech). The low pH elution was mixed with Gerbu adjuvant and used to immunize mice. Each mouse received a subcutaneous injection of 110 μl antigen/adjuvant mix (15 μg antigen+1 μg adjuvant) on[0772]day 0. Mice were subcutaneously boosted as above onday 14 and bled onday 28. Mice were subsequently boosted 1-2 weeks after bleeding and were then bled onday 70.

Anti-[0773]C. botulinumserotype B toxoid titers were determined inday 28 serum from individual mice from each group using the ELISA protocol outlined in Example 29 with the exception that the plates were coated withC. botulinumserotype B toxoid, and the primary antibody was a chicken anti-C. botulinumserotype B toxoid. Seroconversion [relative to control mice immunized with pHisBotE antigen (described below)] was observed with all 9 mice immunized with the purified pHisBotB protein.

The ability of the anti-BotB antibodies to neutralize native[0774]C. botulinumtype B toxin was tested in a mouse-C. botulinumneutralization model using pooled mouse serum (see Ex. 23b). The LD₅₀of purifiedC. botulinumtype B toxin complex (Dr. Eric Johnson, University of Wisconsin, Madison) was determined by a intraperitoneal (IP) method (Schantz and Kautler (1978), supra] using 18-22 g female ICR mice. The amount of neutralizing antibodies present in the serum of the immunized mice was determined using serum antibody titrations. The various serum dilutions (0.01 ml) were mixed with 5 LD₅₀units ofC. botulinumtype B toxin and the mixtures were injected IP into mice. The neutralizations were performed in duplicate. The mice were then observed for signs of botulism for 4 days. Undiluted serum (day 28 or day 70) was found to protect 100% of the injected mice while the 1:10 diluted serum did not. This corresponds to a neutralization titer of 0.05-0.5 IU/ml.

These results demonstrate that seroconversion occurred and neutralizing antibodies were induced when the pHisBotB protein was utilized as the immunogen.[0775]

Example 37Construction of Vectors to Facilitate Expression of His-Tagged BotB Protein in Fermentation Cultures

A number of expression vectors were constructed to facilitate the expression of recombinant BotB protein in large scale fermentation culture. These constructs varied as to the strength of the promoter utilized (T7 or T7lac) and the presence of repressor elements (lacIq) on the plasmid. The resulting constructs varied in the level of expression achieved and in plasmid stability which facilitated the selection of a optimal expression system for fermentation scaleup.[0776]

The BotB expression vectors created for fermentation culture were engineered to utilize the kanamycin rather than the ampicillin resistance gene, and contained either the T7 or T7lac promoter, with or without the lacIq gene for the reasons outlined in Example 30.[0777]

In all cases, the protein expressed by the various expression vectors is the pHisBot B protein described in Example 35, with the only differences between clones being the alteration of various regulatory elements. Using the designations outlined below, the pHisBotB clone (Ex. 35) is equivalent to pHisBotB amp T7lac.[0778]

a) Construction of pHisBotB kan T7lac[0779]

pHisBotB kan T7lac was constructed by insertion of the BglII/HindIII fragment of pHisBotB which contains the BotB gene sequences into the pPA 1870-2680 kan T7lac vector which had been digested with BglII and HindIII (the pPA1870-2680 kan T7lac vector contains the pET24 kan gene in the pET23 vector, such that no lacIq gene is present). Proper construction of pHisBotB kan T7lac was confirmed by restriction digestion.[0780]

b) Construction of pHisBotB kan lacIq T7lac[0781]

pHisBotB kan lacIq T7lac was constructed by insertion of the BglII/HindIII fragment of pHisBotB which contains the BotB gene sequences into similarly cut pET24a vector. Proper construction of pHisBotB kan lacIq T7lac was confirmed by restriction digestion.[0782]

c) Construction of pHisBotB kan lacIq T7[0783]

pHisBotB kan lacIq T7 was constructed by inserting the NdeI/XhoI fragment from pHisBotE kan lacIq T7lac which contains the BotB gene sequences into similarly cleaved pPA1870-2680 kan lacIq T7 vector (this vector contains the T7 promoter, the same N-terminal his-tag as the Bot constructs, the[0784]C. difficiletoxin A insert, and the kan lacIq genes; this cloning replaces theC. difficiletoxin A insert with the BotB insert). Proper construction was confirmed by restriction digestion.

Expression of recombinant BotB protein from these expression vectors and purification of the BotB protein is described in Example 38 below.[0785]

Example 38Fermentation and Purification of Recombinant BotB Protein Utilizing the pHisBotB kan lacIq T7lac, pHisBotB kan T7lac and pHisBotB kan lacIq T7 Vectors

The pHisBotB kan lacIq T7lac, pHisBotB kan T7lac and BotB kan lacIq T7 constructs [all transformed into the B121(DE3) strain] were grown in fermentation cultures to determine the utility of the various constructs for large scale expression and purification of soluble BotB protein. All fermentations were performed as described in Example 31.[0786]

a) Fermentation of pHisBotB kan lacIq T7lac/B121(DE3) Cells[0787]

The fermentation culture was induced 45 min post start of glucose feed with 1 gm IPTG (final concentration=0.4 mM). pH was maintained at 6.5 rather than 7.0. The OD[0788]₆₀₀was 27 at time of induction, then 35, 38, and 40 at 1-3 hrs post induction. Duplicate platings of diluted 1 hr induction samples (dilutions were prepared as described Ex. 31,dilution 3 utilized 3 μl ofdilution 2 cells) on TSA and LB+kan plates yielded 89 TSA colonies and 81 kan colonies (90% kan resistant).

Total and soluble protein extracts were resolved on a 12.5% SDS-PAGE gel and total protein was detected by staining with Coomassie blue. Low level induction of insoluble Bot B protein was observed, increasing from 1 to 3 hrs post induction (no expression was detected in uninduced cells).[0789]

b) Fermentation of pHisBotB kan T7lac/B121(DE3) Cells[0790]

The fermentation culture was induced 1 hr post start of glucose feed with 2 gm IPTG (final concentration=0.8 mM). pH was maintained at 6.5 rather than 7.0. The OD[0791]₆₀₀was 24.5 at time of induction, then 31.5, 32, and 33 at 1-3 hrs post induction, respectively. Duplicate platings of diluted 0 hr and 2 hr induction samples (dilutions were prepared as described Ex. 31;dilution 3 utilized 3 μl ofdilution 2 cells) on TSA and LB+kan plates yielded 32 TSA colonies and 54 kan colonies (all kan resistant) for uninduced cells, and 1 TSA colony and 0kan colonies 2 hr post induction. These results were indicative of strong induction, since viable counts decreased dramatically 2 hrs post induction.

Total and soluble extracts were resolved on a 10% SDS-PAGE gel and total protein was detected by staining with Coomassie blue. Moderate induction of insoluble BotB protein was observed, increasing from 1 to 3 hrs post induction (no expression was detected in uninduced cells).[0792]

c) Fermentation of pHisBotB kan lacIq T7/BU1(DE3) Cells[0793]

The fermentation was induced 2 hr post start of glucose feed with 4 gm IPTG (final concentration=1.6 mM). pH was maintained at 6.5 rather than 7.0. The OD[0794]₆₀₀was 45 at time of induction, then 0.47, 50, and 50 and 55 at 1-4 hrs post induction, respectively. Viable colony counts decreased after induction (96, 1, 1, 2, 3;dilution 3 utilized 31 μl ofdilution 2 cells). Of 63 colonies scored at the time of induction, all 63 retaining the BotB plasmid (kan resistant) and no colonies at induction grew on IPTG+Kan plates (no mutations detected).

Total and soluble extracts were resolved on a 12.5% SDS-PAGE gel and total protein was detected by staining with Coomassie blue. Moderate level induction of insoluble BotB protein was observed, increasing from 1 to 4 hrs post induction (lower level expression was detected in uninduced cells, since the T7 rather than T7lac promoter was utilized).[0795]

d) Purification of pHisBotB Protein From pHisBotB amp T7lac/B121(DE3) Cells[0796]

Soluble recombinant BotB protein was purified utilizing NiNTA resin from 80 ml of cell lysate generated from cells harvested from a pHisBotB fermentation [using the pHisBotB amp T7lac/B121(DE3) strain]. As predicted from the small scale results above, the majority of the induced protein was insoluble. As well, the eluted material was contaminated with multiple[0797]E. colicontaminant proteins. A Coomassie blue-stained SDS-PAGE gel containing extracts derived from pHisBotB amp T7lac/B121(DE3) cells before and during purification is shown in FIG. 34. In FIG. 34,lane 1 contains broad range protein MW markers (BioRad). Lanes 2-5 contain extracts prepared from pHisBotB amp T7lac/B121(DE3) cells grown in fermentation culture;lane 2 contains total protein;lane 3 contains soluble protein;lane 4 contains protein which did not bind to the NiNTA column (i.e., the flow-through) andlane 5 contains protein eluted from the NiNTA column.

The NiNTA alkaline phosphatase conjugate was utilized to detect his-tagged proteins on a Western blot containing total, soluble, soluble (PEI clarified), soluble (after IDA column) and elution samples from the IDA column purification. The results demonstrated that a small percentage of BotB protein was soluble, that the soluble protein was not precipitated by PEI treatment and was quantitatively bound by the IDA column. Since a 1 liter fermentation harvest yielded a 67.5 gm cell pellet, this indicated that the yield of soluble affinity purified BotB protein from the IDA column was 14 mg/liter.[0799]

Example 39Co-Expression of Recombinant BotB Proteins and Folding Chaperones in Fermentation Cultures

Fermentations were performed to determine if the simultaneous overexpression of folding chaperones (i.e., the Gro operon) and the BotB protein resulted in enhanced solubility of the Bot B protein. This example involved fermentation of the pHisBotBkan lacIq T7lac/pACYCGro BL21(DE3), pHisBotB kan T7lac/pACYCGro B121(DE3) and pHisBotBkan lacIq T7/pACYCGro BL21(DE3) cell lines. Fermentation was carried out as described in Example 31; 34 μg/ml chloramphenicol was included in the feeder and fermentation cultures.[0800]

a) Fermentation of pHisBotBkan lacIq T7lac/pACYCGro BL21(DE3) Cells[0801]

Induction was with 4 gms IPTG at 1 hr 15 min post initiation of the glucose feed. The OD[0802]₆₀₀was 38 at time of induction, then 50, 58.5, 62 and 68 at 1-4 hrs post induction. Viable colony counts decreased during induction (24, 0, 0, 2, 0 at 0-4 hr induction;dilution 3 utilized 3 μl ofdilution 2 cells). Of 24 colonies scored at the time of induction, 24 retained the BotB plasmid (kan resistant), 24 contained the chaperone plasmid (chloramphenicol resistant) and no colonies at induction grew on IPTG+Kan plates (no mutations detected).

Total and soluble extracts were resolved on 12.5% SDS-PAGE gels and were either stained with Coomassie blue or subjected to Western blotting (his-tagged proteins were detected utilizing the NiNTA-alkaline phosphatase conjugate). This analysis revealed that the Gro chaperones were induced to high levels, but very low level expression of soluble BotB protein was observed, increasing from 1 to 4.0 hrs post induction (no expression detected in uninduced cells, induced protein detected only on Western blot). The dramatically lower expression of BotB protein in the presence of chaperone may be due to promoter occlusion (i.e. the stronger T7 promoter on the chaperone plasmid was preferentially utilized).[0803]

b) Fermentation of pHisBotB kan T7lac/pACYCGro/B121(DE3) Cells[0804]

Induction was with 4 gms IPTG at 1 hr post initiation of the glucose feed. The OD[0805]₆₀₀was 33.5 at time of induction, then 44, 51, 58.5 and 69 at 14 hrs post induction. Viable colony counts decreased after 2 hrs induction (43, 65, 74, 0 (70), 0 (70) at 0-4 hr induction; bracketed numbers represent microcolonies;dilution 3 utilized 3 μl ofdilution 2 cells). Most colonies at induction retained the BotB plasmid (kan resistant)and the chaperone plasmid (chloramphenicbl resistant) and no colonies at induction grew on IPTG+Kan plates (no mutations detected).

Total and soluble extracts were resolved on a 12.5% SDS-PAGE gel and subjected to Western blotting; his-tagged proteins were detected utilizing the NiNTA-alkaline phosphatase conjugate. This analysis revealed that the Gro chaperones were induced to high levels and low level expression of soluble Bot B protein was observed, increasing from 1 to 4.0 hrs post induction (no expression detected in uninduced cells).[0806]

A small scale IDA purification of BotB protein from a 250 ml PEI clarified 15% w/v extract (37.5 gm cell pellet) yielded approximately 12.5 mg protein, of which approximately 50% was BotB protein and 50% was GroEL chaperone (assessed by Coomassie staining of a 10% SDS-PAGE gel). The NiNTA alkaline phosphatase conjugate was utilized to detect his-tagged proteins on a Western blot containing total, soluble, soluble (PEt clarified), soluble (after IDA column) and elution samples from the IDA column purification. The results demonstrated that all of the BotB protein produced by the pHisBotB kan T7lac/pACYCGro/B121(DE3) cells was soluble; the BotB protein was not precipitated by PEI treatment and was quantitatively bound by the IDA column. Since a 1 liter fermentation harvest yielded a 75 gm cell pellet, this indicated that the yield of soluble affinity purified bot B protein from this fermentation was 12.5 mg/liter. These results also demonstrated that additional purification steps are necessary to separate the chaperone proteins from the BotB protein.[0807]

c) Fermentation of pHisBotBkan lacIq T7/pACYCGro/BL21(DE3) Cells[0808]

Induction was with 4 gms IPTG at 2 hr post initiation of the glucose feed. The OD[0809]₆₀₀was 46 at time of induction, then 56, 63, 69 and 71.5 at 1-4 hrs post induction. Viable colony counts decreased after induction (58, 3(5), 3, 0, 0 at 0-4 hr induction; bracketed numbers represent microcolonies;dilution 3 utilized 3 μl ofdilution 2 cells). All (53/53) colonies scored at the time of induction retained the BotB plasmid (kan resistant) and the chaperone plasmid (chloramphenicol resistant) and no colonies at induction grew on IPTG+Kan plates (no mutations detected).

Total and soluble extracts were resolved on a 10% SDS-PAGE gels and Western blotted and his-tagged proteins were detected utilizing the NiNTA-alkaline phosphatase conjugate. This analysis revealed that the Gro chaperones were induced to high levels (observed by ponceau S staining), and a much higher expression of soluble Bot B protein (compared to expression in the pHisBotB kan T7lac/pACYCGro fermentation) was observed at all timepoints, including uninduced cells (some increase in BotB protein levels were observed after induction).[0810]

A small scale IDA purification of BotB protein from a 100 ml PEI clarified 15% w/v extract (15 gm cell pellet) yielded approximately 40 mg protein, of which approximately 50% was BotB protein and 50% was GroEL chaperone, as assessed by Coomassie staining of a 10% SDS-PAGE gel. The NiNTA alkaline phosphatase conjugate was utilized to detect his-tagged proteins on a Western blot containing total, soluble, soluble (PEI clarified), soluble (after IDA column) and elution samples from the IDA column purification. The results demonstrated that a significant percentage (i.e., ˜10-20%) of BotB protein was soluble, that the solubilized protein was not precipitated by PEI treatment and was quantitatively bound by the IDA column. Since a 10 liter fermentation yielded a 108 gm cell pellet, this indicated that the yield of soluble affinity purified BotB protein from this fermentation was 144 mg/liter.[0811]

In a scale up experiment, 2 liters of a 20% w/v PEI clarified lysate of pHisBotB kan lacIq T7/pACYCGro/BL21(DE3) cells were purified on a large scale IDA column. The purification was performed in duplicate. The total yield of BotB protein was 220 and 325 mgs protein in the two experiments (assuming 1 mg/ml solution=2.0 OD[0812]₂₈₀/ml). This represents 0.7% or 1.0%, respectively, of the total soluble cellular protein (assuming a PET lystate having a concentration of 8 mg protein/ml and that the eluted material comprises a 1:1 mixture of BotB and folding chaperone). The NiNTA alkaline phosphatase conjugate was utilized to detect his-tagged proteins on a Western blot containing total, soluble, soluble (PEI clarified), soluble (after IDA column) and elution samples from the IDA column purification. These results demonstrated that a significant percentage (i.e., ˜10-20%) of the BotB protein was soluble, that the solubilized protein was not precipitated by PEI treatment and was quantitatively bound by the IDA column. Since a 1 liter fermentation harvest yielded a 108 gm cell pellet, this indicated that the yield of soluble affinity purified BotB protein from the large scale purification was 60 mg or 89 mg/liter. These results also demonstrated that further purification would be necessary to remove the contaminating chaperone protein.

The above results provide methodologies for the purification of soluble BotB protein from fermentation cultures, in a form contaminated predominantly with a single[0813]E. coliprotein (the folding chaperone utilized to enhance solubility). In the next example, methods are provided for the removal of the contaminating chaperone protein.

Example 40Removal of Contaminating Folding Chaperone Protein from Purified RecombinantC. botulinumType B Protein

In this example size exclusion chromatography and ultrafiltration was used to purify recombinant BotB protein from the folding chaperones and imidazole in IDA-purified material.[0814]

To enhance the solubility of the recombinant BotB protein during scale-up, the protein was co-expressed with folding chaperones (see Ex. 39). During the Ni-IDA purification step, the folding chaperones co-eluted with the BotB protein in 800 mM imidazole; therefore, a second purification step was required to isolate the BotB free of folding chaperones.[0815]Lane 3 of FIG. 35 contains proteins eluted from an IDA column to which a lysate of pHisBotB kan lacIq T7/pACYCGro/BL21(DE3) cells had been applied; the proteins were resolved on a 4-15% polyacrylamide precast gradient gel (Bio-Rad, Hercules, Calif.) run under native conditions and then stained with Coomassie blue. In FIG. 35,

lanes

1 and 4 contain proteins present inpeak 1 andpeak 2 from a Sephacryl S-100 column run as described below;lane 2 is blank.

As seen in[0816]lane 3 of FIG. 35, the IDA-purified sample consists primarily of the folding chaperones and the BotB protein. The fact that the chaperones and the Bot B antigen appear as two distinct bands under native conditions suggested they were not complexed together and therefore, it should be possible to separate them, using either a gradient of imidazole concentrations or size exclusion methods.

In order to determine whether a gradient of imidazole concentrations could be used to separate the chaperone from the BotB protein, a step gradient using imidazole at 200, 400, 600, and 800 mM in 50 mM sodium phosphate, 0.5 M NaCl and 10% glycerol, pH 6.8 was applied to an IDA column (containing proteins bound from a lysate of pHisBotB kan lacIq T7/pACYCGro/BL21(DE3) cells). By narrowing the range of imidazole concentrations, it was hoped that the BotB and chaperone proteins would differentially elute at different concentrations of imidazole. Eluted proteins were monitored by absorbance at 280 nm and collected either manually or with a fraction collector (BioRad). Protein was found to elute at 200 and 400 mM imidazole only.[0817]

FIG. 36 shows a Coomassie stained SDS-PAGE gel containing protein eluted during the imidazole step gradient.[0818]Lane 1 contains broad range MW markers (BioRad).Lane 2 contains BotB protein purified by IDA chromatography of an extract of pHisBotB/BL21(DE3) pLysS cells grown in shaker flask culture (i.e., no co-expression of chaperones; Ex. 35).Lane 3 contains a 20% w/v PEI clarified lysate of pHisBotB kan lacIq T7/pACYCGro/BL21(DE3) cells (i.e., the lysate prior to purification by IDA chromatography).

Lanes

4 and 5 contain protein which eluted at 200 or 400 mM imidazole, respectively.Lane 6 is blank.

Lanes

7 and 8 contain ⅕ the load present in

lanes

4 and 5.

As shown in FIG. 36, both the chaperone and the BotB protein eluted in 200 mM imidazole, and more chaperone elutes in 400 mM imidazole, however no concentration of imidazole tested permitted the elution of BotB protein alone. Consequently, no significant purification was achieved using imidazole at these concentrations.[0819]

Because of the considerable difference in molecular weights between the folding chaperone, which is a multimer with a total molecular weight around 400 kD (as determined on a Shodex KB 804 sizing column by HPLC), and the recombinant BotB protein (molecular weight around 50 kD), size exclusion chromatography was next examined for the ability to separate these proteins.[0820]

a) Size Exclusion Chromatography[0821]

A column containing Sephacryl S-100 HR(S-100) (Pharmacia) was poured (2.5 cm×24 cm; ˜110 ml bed volume). The column was equilibrated in a buffer consisting of phosphate buffered saline (10 mM potassium phosphate, 150 mM NaCl, pH 7.2) and 10% glycerol (Mallinkrodt). Typically, 5 ml of the IDA-purified BotB protein was filtered through a 0.45μ syringe filter and applied to the column, and the equilibration buffer was pumped through the column at a flow rate of 1 ml/minute. Eluted proteins were monitored by absorbance at 280 nm and collected either manually or with a fraction collector. Appropriate tubes were pooled, if necessary, and the protein was quantitated by absorbance at 280 nm and/or by BCA protein assay. The isolated peaks were then analyzed by native and/or SDS-PAGE to identify the protein and evaluate the purity.[0822]

Because of its larger size, the folding chaperone eluted first, followed by the recombinant BotB protein. A smaller third peak was observed which failed to stain when analyzed by SDS-PAGE and therefore was presumed to be imidazole.[0823]

SDS-PAGE analysis (12.5% polyacrylamide, reduced samples) was used to evaluate the purity of the IDA-purified recombinant BotB protein before and after S-100 purification. The results are shown in FIG. 33.[0824]

In FIG. 33,[0825]lane 1 contains broad range MW markers (BioRad).Lane 5 contains IDA-purified BotB protein.Lane 6 contains IDA-purified BotB protein following S-100 purification.Lane 7 is blank (lanes 2-4 were discussed in Ex. 34 above).

The results shown in FIG. 33 show that the IDA-purified BotB is significantly contaminated with the folding chaperone (molecular weight about 60 kD under reducing conditions; lane 6). Following S-100 purification, the amount of folding chaperone present in the BotB sample was reduced dramatically (lane 7). Visual inspection of the Coomassie stained SDS-PAGE gel revealed that after S-100 purification, >90% of the total protein present was BotB.[0826]

The IDA-purified BotB and the S-100-purified BotB samples were analyzed by HPLC on a size exclusion column (Shodex KB 804); this analysis revealed that the BotB protein represented 64% of the total protein in the IDA-purified sample and that following S-100 purification, the BotB protein represented >95% of the total protein in the sample.[0827]

The IDA-purified BotB material was also applied to a ACA 44 (SpectraPor, Houston, Tex.) column. The ACA 44 resin is equivalent to the S-100 resin and chromatography using the ACA 44 resin was carried out exactly as described above for the S-100 resin. The ACA 44 resin was found to separate the recombinant BotB protein from the folding chaperone. The ACA 44-purified BotB sample was analyzed for endotoxin using the LAL assay (Associates of Cape Cod) as describe in Example 24. Two aliqouts of the ACA 44-purified BotB preparation were analyzed and were found to contain either 58 to 116 EU/mg recombinant protein or 94 to 189 EU/mg recombinant protein.[0828]

These results demonstrate that size exclusion chromatography can be used to purify the recombinant BotB protein from the folding chaperone and imidazole in IDA-purified material.[0829]

b) Ultrafiltration for the Separation of Recombinant BotB Protein and Chaperones[0830]

Ultrafiltration was examined as an alternative method for the separation recombinant BotB protein and folding chaperones in IDA-purified material. While in this example only mixtures of BotB and chaperones were separated by ultrafiltration, this technique is suitable for use with recombinant BotA and BotE proteins as well provided that the wash buffers used are altered as necessary to take into account different requirements for solubility.[0831]

The recombinant BotB protein and folding chaperones were separated using a two-step sequential ultrafiltration method. The first membrane used had a nominal molecular weight cutoff (MWCO) of approximately 100 kD; this membrane retains the larger folding chaperone while allowing the smaller recombinant protein to pass through. The addition of several volumes of wash buffer may be required to efficiently wash the recombinant protein through the membrane. The second step utilized a membrane with a nominal MWCO of approximately 10 kD. During this step, the recombinant antigen was retained by the membrane and could be concentrated to the degree desired and the imidazole and excess wash buffer passed through the membrane.[0832]

Twenty-seven milliliters of an IDA-purified BotB preparation was ultrafiltered through a 47 mm YM 100 (100 kD MWCO) membrane (Amicon) in a 50 ml stirred cell (Amicon). The membrane was washed in dd H[0833]₂O prior to use as recommended by the manufacturer. Six volumes of 10% glycerol in PBS were washed through to remove most of the recombinant BotB protein and this wash was collected in a separate vessel. The resulting BotB protein-rich filtrate was then concentrated 12-fold using a YM 10 (10 kD MWCO) membrane (Amicon), to a final volume of 14 ml. TheYM 100 andYM 10 concentrates were analyzed along with the lysate starting material by native PAGE using a 4-15% pre-cast gradient gel (BioRad). The results are shown in FIG. 37.

In FIG. 37,[0834]lane 1 contains IDA-purified BotB derived from a shaker flask culture (i.e., no co-expression of chaperones; Ex. 35);lane 2 contains a 20% w/v PEI clarified lysate of pHisBotB kan lacIq T7/pACYCGro/BL21(DE3) cells;lane 3 shows the lysate oflane 3 after IDA purification;lane 4 contains theYM 10 concentrate andlane 5 contains theYM 100 concentrate.

The results shown in FIG. 37 demonstrate that the recombinant BotB protein can be purified away from the folding chaperone by ultrafiltration through a 100 kD MWCO membrane (lane 4), leaving the chaperone protein in the 100 kD concentrate (lane 5). Analysis of the sample in[0835]lane 5 also showed that very little of the BotB protein was retained by the 100 kD MWCO membrane after 6 volumes of wash buffer had been applied.

The BotB samples following IDA chromatography and following ultrafiltration through the[0836]YM 100 membrane were anlyzed by HPLC on a size exclusion column (Shodex KB 804); this analysis revealed that the BotB protein represented 64% of the total protein in the IDA-purified sample and that following ultrafiltration through theYM 100 membrane, the BotB protein represented >96% of the total protein in the sample.

The BotB protein purified by ultrafiltration through the[0837]YM 100 membrane was examined for endotoxin using the LAL assay (Associates of Cape Cod) as describe in Example 24. Two aliqouts of the YM 100-purified BotB preparation were analyzed and were found to contain either 18 to 36 EU/mg recombinant protein or 125 to 250 EU/mg recombinant protein.

The above results demonstrate that size exclusion chromatography and ultrafiltration can be used to purify recombinant botulinal toxin proteins away from folding chaperones.[0838]

Example 41Cloning and Expression of the C Fragment of theC. botulinumSerotype E Toxin Gene

The[0839]C. botulinumtype E neurotoxin gene has been cloned and sequenced from several different strains [Poulet et al. (1992) Biochem. Biophys. Res. Commun. 183:107 (strain Beluga); Whelan et al. (1992) Eur. J. Biochem. 204:657 (strain NCTC 11219); Fujii et al. (1990) Microbiol. Immunol. 34:1041 (partial sequence of strains Mashike, Iwani and Otaru) and Fujii et al. (1993) J. Gen. Microbiol. 139:79 (strain Mashike)]. The nucleotide sequence of the type E toxin gene is available from the EMBL sequence data bank under accession numbers X62089 (strain Beluga) and X62683 (strain NCTC 11219). The nucleotide sequence of the coding region (strain Beluga) is listed in SEQ ID NO:49. The amino acid sequence of theC. botulinumtype E neurotoxin derived from strain Belgua is listed in SEQ ID NO:50. The nucleotide sequence of the coding region (strain NCTC 11219) is listed in SEQ ID NO:51. The amino acid sequence of theC. botulinumtype E neurotoxin derived from strain NCTC 11219 is listed in SEQ ID NO:52.

The DNA sequence encoding the native[0840]C. botulinumserotype E C fragment gene derived from the Beluga strain can be expressed as a histidine-tagged protein using the pETHisb vector; the resulting coding region is listed in SEQ ID NO:53 and the corresponding amino acid sequence is listed in SEQ ID NO:54. The DNA sequence encoding the C fragment of the nativeC. botulinumserotype E gene derived from the NCTC 11219 strain can be expressed as a histidine-tagged fusion protein using the pETHisb vector; the resulting coding region is listed in SEQ ID NO:55 and the corresponding amino acid sequence is listed in SEQ ID NO:56. The C fragment region from any strain ofC. botulinumserotype E can be amplified and expressed using the approach illustrated below using the C fragment derived fromC. botulinumtype E 2231strain (ATCC #17786).

The type E neurotoxin gene is synthesized as a single polypeptide chain which may be converted to a double-chain form (i.e., a heavy chain and a light chain) by cleavage with trypsin; unlike the type A neurotoxin, the type E neurotoxin exists essentially only in the single-chain form. The 50 kD carboxy-terminal portion of the heavy chain is referred to as the C fragment or the H[0841]_Cdomain. Expression of the C fragment ofC. botulinumtype E toxin in heterologous hosts (e.g.,E. coli) has not been previously reported.

The native C fragment of the[0842]C. botulinumserotype E toxin (BotE) gene was cloned and inserted into expression vectors to facilitate expression of the recombinant BotE protein inE. coli. This example involved PCR amplification of the gene, cloning, and construction of expression vectors.

The BotE serotype gene was isolated using PCR as described for the BotA serotype gene in Example 28. The[0843]C. botulinumtype E strain was obtained from the American Type Culture Collection (ATCC #17786; strain 2231). The following primer pair was used in the PCR amplification: 5′-CGCCATGGCTCTTTCTTCTTAT ACAGATGAT-3′ (5′ primer, engineered NcoI site underlined) (SEQ ID NO:57) and 5′-GCAAGCTTTTATTTTTCTTGCCATCCATG-3′ (3′ primer, engineered HindIII site underlined, native gene termination codon italicized) (SEQ ID NO:58). The PCR product was inserted into pCRscript as described in Example 28. The resulting pCRscript BotE clone was confirmed by restriction digestion, as well as, by obtaining the sequence of approximately 300 bases located at the 5′ end of the C fragment coding region using standard DNA sequencing methods. The resulting BotE sequence was identical to that of the publishedC. botulinumtype E toxin sequence [Whelan et al (1992), supra].

The NheI(filled)/HindIII fragment from a pCRscript BotE recombinant was cloned into pETHisb vector as described for BotA C fragment in Example 28. The resulting construct was termed pHisBotE. pHisBotE expresses the BotE gene under the control of the T7 lac promoter and the resulting protein contains an N-terminal 10×His-tag affinity tag.[0844]

The pHisBotE expression construct was transformed into BL21(DE3) pLysS competent cells and 1 liter cultures were grown, induced and his-tagged proteins were purified utilizing a NiNTA resin (eluted in low pH elution buffer) as described in Example 28. Total, soluble and purified proteins were resolved by SDS-PAGE and detected by Coomassie staining. The results are shown in FIG. 38.[0845]

In FIG. 38,[0846]lane 1 contains broad range MW markers (BioRad);lane 2 contains a total protein extract;lane 3 contains a soluble protein extract;lane 4 contains proteins present in the flow through from the NiNTA column (this sample was not diluted prior to loading and therefore represents aload 5× that of the load applied for the total and soluble extracts inlanes 2 and 3);lane 5 contains proteins eluted from the NiNTA column;lane 6 contains protein eluted from a NiNTA column which had been stored at −20° C. for 1 year.

The pHisBotE protein was expressed at moderate levels (7 mg/liter) as a totally soluble protein. The purified protein migrated as a single band of the predicted MW.[0847]

Western blot hybridization utilizing a chicken anti-[0848]C. botulinumserotype E toxoid primary antibody (generated by immunization of hens as described in Example 3 usingC. botulinumserotype E toxoid) was also performed on the total, soluble and purified BotE proteins. Samples of BotA and BotB C fragments were also included on the gels to facilitate MW and immunogenicity comparisons. Strong immunoreactivity was detected using the anti-C. botulinumtype E toxoid antibody only with the BotE protein.

These results demonstrate that the native BotE gene sequences can be expressed as a soluble his-tagged protein in[0849]E. coliand purified by metal-chelation affinity chromatography.

Example 42Generation of Neutralizing Antibodies Using the Recombinant pHisBotE Protein

The ability of the purified pHisBotE protein to generate neutralizing antibodies was examined. Nine BALBc mice were immunized with BotE protein (purified as described in Ex. 41) using Gerbu GMDP adjuvant (CC Biotech). The low pH elution was mixed with Gerbu adjuvant and used to immunize mice. Each mouse received a subcutaneous injection of 100 μl antigen/adjuvant mix (35 μg antigen+1 μg adjuvant) on[0850]day 0. Mice were subcutaneously boosted as above onday 14 and bled onday 28. Mice were subsequently boosted and bled onday 70.

Anti-[0851]C. botulinumserotype E toxoid titers were determined inday 28 serum from individual mice from each group using the ELISA protocol outlined in Example 29 with the exception that the plates were coated withC. botulinumserotype E toxoid, and the primary antibody was a chicken anti-C. botulinumserotype E toxoid. Seroconversion [relative to control mice immunized with the p6xHisBotA antigen (Ex. 29)] was observed with all 9 mice immunized with the purified pHisBotE protein.

The ability of the anti-BotE antibodies to neutralize native[0852]C. botulinumtype E toxin was tested in a mouse-C. botulinumneutralization model using pooled mouse serum (see Ex. 23b). The LD₅₀of purifiedC. botulinumtype E toxin complex (Dr. Eric Johnson, University of Wisconsin, Madison) was determined by a intraperitoneal (IP) method [Schantz and Kautler (1978), supra] using 18-22 g female ICR mice. The amount of neutralizing antibodies present in the serum of the immunized mice was determined using serum antibody titrations. The various serum dilutions (0.01 ml) were mixed with 5 LD₅₀units ofC. botulinumtype E toxin and the mixtures were injected [P into mice. The neutralizations were performed in duplicate. The mice were then observed for signs of botulism for 4 days. Undiluted serum fromday 28 did not protect, while undiluted, 1/10 diluted and 1/100diluted day 70 serum protected (1005 of animals) while 1/1000 dilutedday 70 serum did not. This corresponds to a neutralization titer of 50-500 IU/ml.

These results demonstrate that seroconversion occurred and neutralizing antibodies were induced when the recombinant BotE protein was utilized as the immunogen.[0853]

Example 43Construction of Vectors to Facilitate Expression of His-Tagged BotE Protein in Fermentation Cultures

A number of expression vectors were constructed to facilitate the expression of recombinant BotE protein in large scale fermentation culture. These constructs varied as to the strength of the promoter utilized (T7 or T7lac) and the presence of repressor elements (lacIq) on the plasmid. The resulting constructs varied in the level of expression achieved and in plasmid stability which facilitated the selection of a optimal expression system for fermentation scaleup. This example involved a) construction of BotE expression vectors and b) determination of expression levels in small scale cultures.[0854]

a) Construction of BotE Expression Vectors[0855]

The BotE expression vectors created for fermentation culture were engineered to utilize the kanamycin rather than the ampicillin resistance gene, and contained either the T7 or T7lac promoter, with or without the lacIq gene for the reasons outlined in Example 30.[0856]

In all cases, the protein expressed by the various expression vectors is the pHisBotE protein described in Example 41, with the only differences between clones being the alteration of various regulatory elements. Using the designations outlined below, the pHisBotE clone (Ex. 41) is equivalent to pHisBotE amp T7lac.[0857]

i) Construction of pHisBotE kan lacIq T7lac[0858]

pHisBotE kan lacIq T7lac was constructed by inserting the XbaI/HindIII fragment of pHisBotE which contains the BotE gene sequences into XbaI/HindIII-cleaved pET24a vector. Proper construction was confirmed by restriction digestion.[0859]

ii) Construction of pHisBotE kan T7[0860]

pHisBotE kan T7 was constructed by ligating the BotE-containing XbaI/SapI fragment of pHisBotE kan lacIqT7lac to the T7 promoter-containing XbaI/SapI fragment of pET23a. Proper construction was confirmed by restriction digestion.[0861]

iii) Construction of pHisBotE kan lacIqT7[0862]

pHisBotE kan lacIqT7 was constructed by inserting the BglII/HindIII fragment from pHisBotE kan T7 which contains the BotE gene sequences into BglII/HindIII-cleaved pET24 vector. Proper construction was confirmed by restriction digestion.[0863]

b) Determination of BotE Expression Levels in Small Scale Cultures[0864]

The three BotE kan expression vectors described above were transformed into B121(DE3) competent cells and 50 ml (2XYT+40 μg/ml kan) cultures were grown and induced with ITPG as described in Example 28. Total and soluble protein extracts from before and after induction made as described in Example 28. The total and soluble extracts were resolved on a 12.5% SDS-PAGE gel, and his-tagged proteins were detected on a Western blot utilizing the NiNTA-alkaline phosphatase conjugate as described in Example 31(c)(iii). The results showed that all three BotE cell lines expressed his-tagged proteins of the predicted MW for the BotE protein upon induction. The results also demonstrated that the two constructs that contained the T7 promoter expressed the BotE protein before induction, while the T7lac promoter construct did not. Upon induction, the T7 promoter-containing constructs induced to higher levels than the T7lac-containing construct, with the pHisBotE kan lacIqT7/B121(DE3) cells accumulating the maximal levels of BotE protein.[0865]

Example 44Expression and Purification of pHisBotE from Fermentation Cultures

Based on the small scale inductions performed in Example 43, the pHisBotE kan lacIq T7/B121(DE3) strain was selected for fermentation scaleup. This example involved the fermentation and purification of recombinant BotE C fragment protein.[0866]

A fermentation with the pHisBotE kan lacIq T7/B121(DE3) strain was performed as described in Example 31. The fermentation culture was induced 2 hrs post start of the glucose feed with 4 gm IPTG (final concentration=1.6 mM). The OD[0867]₆₀₀was 42 at time of induction, then 46.5, 48, 53 and 54 at 1-4 hrs post induction. Viable colony counts decreased from 0-4 hr induction [131, 4 (28), 7 (3), 7, 8;dilution 3 utilized 6 μl ofdilution 2 cells; bracketed colonies are microcolonies]. All (32/32) colonies scored at the time of induction retained the BotE plasmid (kan resistant) and no colonies at induction grew on IPTG+Kan plates (no mutations detected). These results were indicative of strong promoter induction, since colony viability reduced after induction, and the culture stopped growing during fermentation (stopped at 54 OD₆₀₀/ml).

Total and soluble extracts were resolved on a 12.5% SDS-PAGE gel and total protein was detected by staining with Coomassie blue. The results are shown in FIG. 39.[0868]

In FIG. 39,[0869]lane 1 contains total protein from a pHisBotA kan T7 lac/B121(DE3) pLysS fermentation (Ex. 24). Lanes 2-9 contain extracts prepared from the above pHisBotE kan lacIq T7/B121(DE3) fermentation; lanes 2-4 contain total protein extracts prepared at 0, 1 and 2 hours post-induction, respectively.Lane 5 contains a soluble protein extract prepared at 2 hours post-induction.

Lanes

6 and 7 contain total and soluble extracts prepared at 3 hours post-induction, respectively.

Lanes

8 and 9 contain total and soluble extracts prepared at 4 hours post-induction, respectively.Lane 10 contains broad range MW markers (BioRad).

The results shown in FIG. 39 demonstrate that moderate level induction of totally soluble Bot E protein was observed, increasing from 1 to 4 hrs post induction (no expression was detected in uninduced cells). From a 2 liter fermentation harvest a 155 gm (wet wt) cell pellet was obtained and used to make a PEI-clarified lysate (1 liter in CRB, pH 6.8). The lysate was applied to a large scale IDA column and 200 mg of BotE protein, which was found to be greater than 95% pure (as judged by visual inspection of a Coomassie stained SDS-PAGE gel), was recovered. This represents 2.5% of the total soluble cellular protein (assuming a PEI lysate having a concentration of 8 mg protein/ml) and corresponds to a yield of 100 mg BotE protein/liter of fermentation culture.[0870]

The above results demonstrate that high levels of the recombinant BotE protein can be expressed and purified from fermentation cultures.[0871]

Example 45Removal of Imidazole from Purified Recombinant BotE Protein Preparations

The expression of recombinant BotE protein, unlike the BotA and BotB proteins, did not require the presence of folding chaperones to maintain solubility during scale-up. A size exclusion chromatography step was included however to remove the imidazole from the sample and exchange the IDA elution buffer for one consistent with the BotA antigen.[0872]

A Sephacryl S-100 HR(S-100; Pharmacia) column was poured (2.5 cm×24 cm; bed volume ˜110 ml). Under these conditions, the BotE protein should be retained by the beads to a lesser degree than the smaller imidazole, therefore the BotE protein should elute from the column before the imidazole. The column was equilibrated in a buffer consisting of 50 mM sodium phosphate, 0.5 M NaCl, and 10% glycerol (all reagents from Mallinkrodt). Five milliliters of the IDA-purified BotE protein (Ex. 44) was filtered through a 0.45μ syringe filter and applied to the S-100 column, and equilibration buffer was pumped through the column at a flow rate of 1 ml/minute. Eluted proteins were monitored by absorbance at 280 nm, and collected either manually or with a fraction collector. Appropriate tubes were pooled if necessary, and the protein was quantitated by absorbance at 280 nm and/or BCA protein assay. The isolated peaks were then analyzed by native and/or SDS-PAGE to identify the protein(s) and evaluate the purity.[0873]

FIG. 40 provides a representative chromatogram showing the purification of IDA-purified BotE on the S-100 column. Even though folding chaperones were not over-expressed with this antigen, a small amount of protein eluted at a time consistent with the folding chaperones expressed with BotA and BotB proteins (Gro) (see the first peak). The second peak in the chromatogram contained the BotE protein, and the third peak was presumably imidazole. This presumed imidazole peak was isolated in comparable levels in IDA-purified BotA and BotB protein preparations as well.[0874]

These results demonstrate that size exclusion chromatography can be used to remove imidazole and traces of contaminating high molecular weight proteins from IDA-purified BotE protein preparations.[0875]

The S-100-purified BotE protein was tested for endotoxin contamination using the LAL assay as described in Example 24. This preparation was found to contain 64 to 128 EU/mg recombinant protein and is therefore substantially free of endotoxin.[0876]

The S-100 purified BotE was mixed with purified preparations of BoLA and BotB proteins and used to immunize mice; 5 μg of each Bot protein was used per immunization and alum was included as an adjuvant. After two immunizations with this trivalent vaccine, the immunized mice were challanged with[0877]C. botulinumtoxin. The immunized mice contained neutralizing antibodies sufficient to neutralize between 100,000 to 1,000,000 LD₅₀of either toxin A or toxin B and between 1,000 to 10,000 LD₅₀of toxin E. The titer of neutralizing antibodies directed against toxin E would be expected to increase following subsequent boosts with the vaccine. These results demonstrate that a trivalent vaccine containing recombinant BotA, BotB and BotE proteins provokes neutralizing antibodies.

Example 46Expression of the C Fragment of theC. botulinumSerotype C Toxin Gene and Generation of Neutralizing Antibodies

The[0878]C. botulinumtype C1 neurotoxin gene has been cloned and sequenced [Kimura et al. (1990) Biochem. Biophys. Res. Comm. 171:1304]. The nucleotide sequence of the toxin gene derived from theC. botulinumtype C strain C-Stockholm is available from the EMBL/GenBank sequence data banks under the accession number D90210; the nucleotide sequence of the coding region is listed in SEQ ID NO:59. The amino acid sequence of theC. botulinumtype C1 neurotoxin derived from this strain is listed in SEQ ID NO:60.

The DNA sequence encoding the native[0879]C. botulinumserotype C1-C fragment gene derived from the C-Stockholm strain can be expressed using the pETHisb vector; the resulting coding region is listed in SEQ ID NO:61 and the corresponding amino acid sequence is listed in SEQ ID NO:62. The C fragment region from any strain ofC. botulinumserotype C can be amplified and expressed using the approach illustrated below using the C fragment derived fromC. botulinumtype C C-Stockholm strain. Expression of the C fragment ofC. botulinumtype C1 toxin in heterologous hosts (e.g.,E. coli) has not been previously reported.

The C fragment of the[0880]C. botulinumserotype C1 (BotC1) toxin gene is cloned using the protocols and conditions described in Example 28 for the isolation of the native BotA gene. A number ofC. botulinumserotype C strains (expressing either or both C1 and C2 toxin) are available from the ATCC [e.g., 2220 (ATCC 17782), 2239 (ATCC 17783), 2223 (ATCC 17784; a type C-P strain; C-β strains produce C2 toxin), 662 (ATCC 17849; a type C-α strain; C-α strains produce mainly C1 toxin and a small amount of C2 toxin), 2021 (ATCC 17850; a type C-α strain) and VPI 3803 (ATCC 25766)]. Alternatively, other type C strains may be employed for the isolation of sequences encoding the C fragment ofC. botulinumserotype C toxin.

The following primer pair is used to amplify the BotC gene: 5′-CGCCATGGC TTTATTAAAAGATATAATTAATG-3′ [5′ primer, engineered NcoI site underlined (SEQ ID NO:63)] and 5′-GCAAGCTT7TATTCACTTACAGGTAC AAAACC-3′ [3′ primer, engineered HindIII site underlined, native gene termination codon italicized (SEQ ID NO:64)]. Following PCR amplification, the PCR product is inserted into the pCRscript vector and then the 1.5 kb fragment is cloned into pETHisb vector as described for BotA C fragment gene in Example 28. The resulting construct is termed pHisBotC. Proper construction is confirmed by DNA sequencing of the BotC sequences contained within pHisBotC. pHisBotC expresses the BotC gene sequences under the transcriptional control of the T7 lac promoter and the resulting protein contains an N-terminal 10×His-tag affinity tag. The pHisBotC expression construct is transformed into BL21(DE3) pLysS competent cells and 1 liter cultures are grown, induced and his-tagged proteins are purified utilizing a NiNTA resin (eluted in 250 mM imidazole, 20% glycerol) as described in Example 28. Total, soluble and purified proteins are resolved by SDS-PAGE and detected by Coomassie staining and Western blot hybridization utilizing a Ni-NTA-alkaline phosphatase conjugate (Qiagen) which recognizes his-tagged proteins as described in Example 31 (c)(iii). This analysis permits the determination of expression levels of the pHisBotC protein (i.e., number of mg/liter expressed as a soluble protein). The purified BotC protein will migrate as a single band of the predicted MW (i.e., −50 kD).[0881]

The level of expression of the pHisBotC protein may be modified (increased) by substitution of the T7 promoter for the T7lac promoter, or by inclusion of the lacIq gene on the expression plasmid, and plasmid expressed in BL21(DE3) cell lines in fermentation cultures as described in Example 30. If only very low levels (i.e., less than 0.5%) of soluble pHisBotC protein are expressed using the above expression systems, the pHisBotC construct may be co-expressed with pACYCGro construct as described in Example 32. In this case, the recombinant BotC protein may co-purify with the folding chaperones. The contaminating chaperones may be removed as described in Example 34. Preparations of purified pHisBotC protein are tested for endotoxin contamination using the LAL assay as described in Example 24.[0882]

The purifed pHisBotC protein is used to generate neutralizing antibodies. BALBc mice are immunized with the BotC protein using Gerbu GMDP adjuvant (CC Biotech) as described in Example 36. The ability of the anti-BotC antibodies to neutralize native[0883]C. botulinumtype C toxin is demonstrated using the mouse-C. botulinumneutralization model described in Example 36.

Example 47Expression of the C Fragment of theC. botulinumSerotype D Toxin Gene and Generation of Neutralizing Antibodies

The[0884]C. botulinumtype D neurotoxin gene has been cloned and sequenced [Sunagawa et al. (1992) J. Vet. Med. Sci. 54:905 and Binz et al. (1990) Nucleic Acids Res. 18:5556]. The nucleotide sequence of the toxin gene derived from the CB16 strain is available from the EMBL/GenBank sequence data banks under the accession number S49407; the nucleotide sequence of the coding region is listed in SEQ ID NO:65. The amino acid sequence of theC. botulinumtype D neurotoxin derived from the CB16 strain is listed in SEQ ID NO:66.

The DNA sequence encoding the native[0885]C. botulinumserotype D C fragment gene derived from a BotD expressing strain can be expressed using the pETHisb vector; the resulting coding region is listed in SEQ ID NO:67 and the corresponding amino acid sequence is listed in SEQ ID NO:68. The C fragment region from any strain ofC. botulinumserotype D can be amplified and expressed using the approach illustrated below using the C fragment derived fromC. botulinumtype D CB 16 strain. Expression of the C fragment ofC. botulinumtype D toxin in heterologous hosts (e.g.,E. coli) has not been previously reported.

The C fragment of the[0886]C. botulinumserotype D (BotD) toxin gene is cloned using the protocols and conditions described in Example 28 for the isolation of the native BotA gene. A number ofC. botulinumtype D strains are available from the ATCC [e.g., ATCC 9633, 2023 (ATCC 17851), and VPI 5995 (ATCC 27517)].

The following primer pair is used to amplify the BotD gene: 5′-CGCCATGGC TTTATTAAAAGATATAATTAATG-3′ [5′ primer, engineered NcoI site underlined (SEQ ID NO:63)] and 5′-GCAAGCTT7TACTCTACCCATCCTGGATCCCT-3′ [3′ primer, engineered HindIII site underlined, native gene termination codon italicized (SEQ ID NO:69)]. Following PCR amplification, the PCR product is inserted into the pCRscript vector and then the 1.5 kb fragment is cloned into pETHisb vector as described for BotA C fragment gene in Example 28. The resulting construct is termed pHisBotD.[0887]

pHisBotD expresses the BotD gene sequences under the transcriptional control of the T7 lac promoter and the resulting protein contains an N-terminal 10×His-tag affinity tag. The pHisBotD expression construct is transformed into BL21(DE3) pLysS competent cells and 1 liter cultures are grown, induced and his-tagged proteins are purified utilizing a NiNTA resin as described in Example 28. Total, soluble and purified proteins are resolved by SDS-PAGE and detected by Coomassie staining and Western blot hybridization utilizing a Ni-NTA-alkaline phosphatase conjugate (Qiagen) which recognizes his-tagged proteins as described in Example 31(c)(iii). This analysis permits the determination of expression levels of the pHisBotD protein (i.e., number of mg/liter expressed as a soluble protein). The purified BotD protein will migrate as a single band of the predicted MW (i.e., ˜50 kD).[0888]

The level of expression of the pHisBotD protein may be modified (increased) by substitution of the T7 promoter for the T71ac promoter, or by inclusion of the lacIq gene on the expression plasmid, and plasmid expressed in BL21(DE3) cell lines in fermentation cultures as described in Example 30. If only very low levels (i.e., less than about 0.5%) of soluble pHisBotD protein are expressed using the above expression systems, the pHisBotD construct may be co-expressed with pACYCGro construct as described in Example 32. In this case, the recombinant BotD protein may co-purify with the folding chaperones. The contaminating chaperones may be removed as described in Example 34. Preparations of purified pHisBotD protein are tested for endotoxin contamination using the LAL assay as described in Example 24.[0889]

The purifed pHisBotD protein is used to generate neutralizing antibodies. BALBc mice are immunized with the BotD protein using Gerbu GMDP adjuvant (CC Biotech) as described in Example 36. The ability of the anti-BotD antibodies to neutralize native[0890]C. botulinumtype D toxin is demonstrated using the mouse-C. botulinumneutralization model described in Example 36.

Example 48Expression of the C Fragment of theC. botulinumSerotype F Toxin Gene and Generation of Neutralizing Antibodies

The[0891]C. botulinumtype F neurotoxin gene has been cloned and sequenced [East et al. (1992) FEMS Microbiol. Lett. 96:225]. The nucleotide sequence of the toxin gene derived from the 202F strain (ATCC 23387) is available from the EMBL/GerBank sequence data banks under the accession number M92906; the nucleotide sequence of the coding region is listed in SEQ ID NO:70. The amino acid sequence of theC. botulinumtype F neurotoxin derived from the 202F strain is listed in SEQ ID NO:71.

The DNA sequence encoding the native[0892]C. botulinumserotype F C fragment gene derived from the 202F strain can be expressed using the pETHisb vector; the resulting coding region is listed in SEQ ID NO:72 and the corresponding amino acid sequence is listed in SEQ ID NO:73. The C fragment region from any strain ofC. botulinumserotype F can be amplified and expressed using the approach illustrated below using the C fragment derived fromC. botulinumtype F 202F strain. Expression of the C fragment ofC. botulinumtype F toxin in heterologous hosts (e.g.,E. coli) has not been previously reported.

The C fragment of the[0893]C. botulinumserotype F (BotF) toxin gene is cloned using the protocols and conditions described in Example 28 for the isolation of the native BotA gene. TheC. botulinumtype F 202F strain is obtained from the American Type Culture Collection (ATCC 23387). Alternatively, sequences encoding the BotF toxin may be isolated from any BotF expressing strain [e.g., VPI 4404 (ATCC 25764), VPI 2382 (ATCC 27321) and Langeland (ATCC 35415)].

The following primer pair is used to amplify the BotF gene: 5′-CGCCATGGC TATTCTAATTATATATTTTAATAG-3′ [5′ primer, engineered NcoI site underlined (SEQ ID NO:74)] and 5′-GCAAGCTTTCATTCTTTCCATCCATTCTC-3′ [3′ primer, engineered HindIII site underlined, native gene termination codon italicized (SEQ ID NO:75)]. Following PCR amplification, the PCR product is inserted into the pCRscript vector and then the 1.5 kb fragment is cloned into pETHisb vector as described for BotA C fragment gene in Example 28. The resulting construct is termed pHisBotF.[0894]

pHisBotF expresses the BotF gene sequences under the transcriptional control of the T7 lac promoter and the resulting protein contains an N-terminal 10XHis-tag affinity tag. The pHisBotF expression construct is transformed into BL21(DE3) pLysS competent cells and 1 liter cultures are grown, induced and his-tagged proteins are purified utilizing a NiNTA resin as described in Example 28. Total, soluble and purified proteins are resolved by SDS-PAGE and detected by Coomassie staining and Western blot hybridization utilizing a Ni-NTA-alkaline phosphatase conjugate (Qiagen) which recognizes his-tagged proteins as described in Example 31(c)(iii). This analysis permits the determination of expression levels of the pHisBotF protein (i.e., number of mg/liter expressed as a soluble protein). The purified BotF protein will migrate as a single band of the predicted MW (i.e., ˜50 kD).[0895]

The level of expression of the pHisBotF protein may be modified (increased) by substitution of the T7 promoter for the T7lac promoter, or by inclusion of the lacIq gene on the expression plasmid, and plasmid expressed in BL21(DE3) cell lines in fermentation cultures as described in Example 30. If only very low levels (i.e., less than about 0.5%) of soluble pHisBotF protein are expressed using the above expression systems, the pHisBotF construct may be co-expressed with pACYCGro construct as described in Example 32. In this case, the recombinant BotF protein may co-purify with the folding chaperones. The contaminating chaperones may be removed as described in Example 34. Preparations of purified pHisBotF protein are tested for endotoxin contamination using the LAL assay as described in Example 24.[0896]

The purifed pHisBotF protein is used to generate neutralizing antibodies. BALBc mice are immunized with the BotF protein using Gerbu GMDP adjuvant (CC Biotech) as described in Example 36. The ability of the anti-BotF antibodies to neutralize native[0897]C. botulinumtype F toxin is demonstrated using the mouse-C. botulinumneutralization model described in Example 36.

Example 49Expression of the C Fragment of theC. botulinumSerotype G Toxin Gene and Generation of Neutralizing Antibodies

The[0898]C. botulinumtype G neurotoxin gene has been cloned and sequenced (Campbell et al. (1993) Biochimica et Biophysica Acta 1216:487 and Binz et al. (1990) Nucleic Acids Res. 18:5556]. The nucleotide sequence of the toxin gene derived from the 113/30 strain (NCFB 3012) is available from the EMBL/GenBank sequence data banks under the accession number X74162; the nucleotide sequence of the coding region is listed in SEQ ID NO:76. The amino acid sequence of theC. botulinumtype G neurotoxin derived from this strain is listed in SEQ ID NO:77.

The DNA sequence encoding the native[0899]C. botulinumserotype G C fragment gene derived from the 113/30 strain can be expressed using the pETHisb vector; the resulting coding region is listed in SEQ ID NO:78 and the corresponding amino acid sequence is listed in SEQ ID NO:79. The C fragment region from any strain ofC. botulinumserotype G can be amplified and expressed using the approach illustrated below using the C fragment derived fromC. botulinumtype G 113/30 strain. Expression of the C fragment ofC. botulinumtype G toxin in heterologous hosts (e.g.,E. coli) has not been previously reported.

The C fragment of the[0900]C. botulinumserotype G (BotG) toxin gene is cloned using the protocols and conditions described in Example 28 for the isolation of the native BotA gene. TheC. botulinumtype G 113/30 strain is obtained from the NCFB. The following primer pair is used to amplify the BotG gene: 5′-CGCCATGGCTGAC ACAATTTTAATACA AGT-3′ [5′ primer, engineered NcoI site underlined (SEQ ID NO:80)] and 5′-GCCTCGAGTTATTCTGTCCATCCTTCATCCAC-3′ [3′ primer, engineered XhoI site underlined, native gene termination codon italicized (SEQ ID NO:81)]. Following PCR amplification, the PCR product is inserted into the pCRscript vector and then the 1.5 kb fragment is cloned into pETHisb vector as described for BotA C fragment gene in Example 28 with the exception that the sequences encoding BotG are excised from the pCRscript vector by digestion with NcoI and XhoI and the NcoI site is blunted (the BotG sequences contain an internal HindIII site). This NcoI(filled)/XhoI fragment is then ligated to the pETHisb vector which has been digested with NheI and SalI and the NheI site is blunted. The resulting construct is termed pHisBotG. pHisBotG expresses the BotG gene sequences under the transcriptional control of the T7 lac promoter and the resulting protein contains an N-terminal 10×His-tag affinity tag. The pHisBotG expression construct is transformed into BL21(DE3) pLysS competent cells and 1 liter cultures are grown, induced and his-tagged proteins are purified utilizing a NiNTA resin as described in Example 28. Total, soluble and purified proteins are resolved by SDS-PAGE and detected by Coomassie staining and Western blot hybridization utilizing a Ni-NTA-alkaline phosphatase conjugate (Qiagen) which recognizes his-tagged proteins as described in Example 31 (c)(iii). This analysis permits the determination of expression levels of the pHisBotG protein (i.e., number of mg/liter expressed as a soluble protein). The purified BotG protein will migrate as a single band of the predicted MW (i.e., ˜50 kD).

The level of expression of the pHisBotG protein may be modified (increased) by substitution of the T7 promoter for the T7lac promoter, or by inclusion of the lacIq gene on the expression plasmid, and plasmid expressed in BL21(DE3) cell lines in fermentation cultures as described in Example 30. If only very low levels (i.e., less than about 0.5%) of soluble pHisBotG protein are expressed using the above expression systems, the pHisBotG construct may be co-expressed with pACYCGro construct as described in Example 32. In this case, the recombinant BotG protein may co-purify with the folding chaperones. The contaminating chaperones may be removed as described in Example 34. Preparations of purified pHisBotG protein are tested for endotoxin contamination using the LAL assay as described in Example 24.[0901]

The purifed pHisBotG protein is used to generate neutralizing antibodies. BALBc mice are immunized with the BotG protein using Gerbu GMDP adjuvant (CC Biotech) as described in Example 36. The ability of the anti-BotG antibodies to neutralize native[0902]C. botulinumtype G toxin is demonstrated using the mouse-C. botulinumneutralization model described in Example 36.

Example 50Expression of Recombinant Botulinal Toxin Proteins in Eucaryotic Host Cells

Recombinant botulinal C fragment proteins may be expressed in eucaryotic host cells, such as yeast and insect cells.[0903]

a) Expression in Yeast[0904]

Botulinal C fragments derived from serotypes A, B, C, D, E, F and G may be expressed in yeast cells using a variety of commercially available vectors. For example, the pPIC3K and pPIC9K expression vectors (Invitrogen) may be employed for expression in the methylotrophic yeast,[0905]Pichia pastoris. When the pPIC3K vector is employed, expression of the botulinal C fragment protein will be intracellular. When the pPIC3K vector is employed, the botulinal C fragment protein will be secreted (the alpha factor secretion signal is provided on the pPIC9K vector).

DNA sequences encoding the desired C fragment is inserted into these vectors using techniques known to the art. Briefly, the desired botulinal expression cassette (including sequences encoding the his-tag; described in the preceding examples) is amplified using the PCR in conjunction with primers that incorporate unique restriction sites at the termini of the amplified fragment. Suitable restriction enzyme sites include SnaBi, EcoRI, AvrII and NotI. When the botulinal C fragment is to be expressed using the pPIC3K vector, the initiator methionine (ATG) is provided by the desired Bot gene sequence and a Kozak consensus sequence is engineered upstream of the ATG (e.g., ACCATGG).[0906]

The amplified restriction fragment containing the botulinal C fragment gene is then cloned into the desired expression vector. Recombinant clones are integrated into the[0907]Pichia pastorisgenome and recombinant protein expression is induced using methanol following the manufacturer's instructions (Invitrogen Pichia expression kit manual).

[0908]C. botulinumgenes are A/T rich and contain multiple sequences that are similar to yeast transcriptional termination signals (e.g., TTTTTATA). If premature transcription termination is observed when the botulinal C fragment genes are expressed in yeast, the transcription termination signals present in the C fragment genes can be removed by either site directed mutagenesis (utilizing the PALTER system; Promega) or by construction of synthetic genes utilizing overlapping synthetic primers.

The botulinal C fragment genes may be expressed in other yeast cells using other commercially available vectors [e.g., using the pYES2 vector (Invitrogen) and[0909]S. cerevisiaecells (Invitrogen)].

b) Expression in Insect Cells[0910]

Botulinal C fragments derived from serotypes A, B, C, D, E, F and G may be expressed in insect cells using a variety of commercially available vectors. For example, the pBlueBac4 transfer vector (Invitrogen) may be employed for expression in[0911]Spodoptera frugiperda(Sf9) insect cells (baculovirus expression system) (equivalent baculovirus vectors and host cells are avaialble from other vendors, e.g., Pharmingen, San Diego, Calif.). Botulinal C fragments contained on NcoI/HindIII fragments contained within the pHisBotA-G expression constructs (described in the preceding examples) are cloned into the pBlueBac4 vector (digested with NcoI and HindIII); the NcoI site present on the C fragment constructs overlaps with the start codon of the fusion proteins. In the case of botulinal C fragment clones that contain internal HindIII sites (e.g., using the BotG sequences described in Ex. 49), the C fragment gene is contained within a NcoI/XhoI fragment on the pHisBot construct. This NcoI/XhoI fragment is excised from pHisBot and inserted into pBlueBac4 digested with NcoI and SalI. Recombinant baculoviruses are made and the desired recombinant C fragment is expressed in Sf9 cells using the protocols provided by the manufacturer (Invitrogen MaxBac manual). The resulting constructs will express the pHisBot protein intracellularly (including the N-terminal his-tag) under the control of the polyhedrin promoter. For extracellular secretion of botulinal C fragment proteins, the C fragment sequences from the pHisBot constructs are cloned into the pMelBacB vector (Invitrogen) as described above for the pBlueBac4 vector. When the pMelBacB vector is employed, the his-tagged botulinal C fragment proteins are secreted (utilizing a vector-encoded honeybee melittin secretion signal) and contain a nine amino acid extension at the N-terminus.

His-tagaed botulinal C fragments expressed in yeast or insect cells are purified using metal chelation columns as described in the preceding examples.[0912]

From the above it is clear that the present invention provides compositions and methods for the preparation of effective multivalent vaccines against[0913]C. botulinumneurotoxin. It is also contemplated that the recombinant botulinal proteins be used for the production of antitoxins. All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention.

1 GGAAATTTAG CTGCAGCATC TGAC 24

2 TCTAGCAAAT TCGCTTGTGT TGAA 24

3 CTCGCATATA GCATTAGACC 20

4 CTATCTAGGC CTAAAGTAT 19

8 Ser Tyr Lys Ile Ile Asn Gly Lys His Phe Tyr Phe Asn Asn Asp Gly 1 5 10 15 Val Met Gln Leu Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr Phe 20 25 30 Ala Pro Ala Asn Thr Gln Asn Asn Asn Ile Glu Gly Gln Ala Ile Val 35 40 45 Tyr Gln Ser Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Asp 50 55 60 Asn Asn Ser Lys Ala Val Thr Gly Trp Arg Ile Ile Asn Asn Glu Lys 65 70 75 80 Tyr Tyr Phe Asn Pro Asn Asn Ala Ile Ala Ala 85 90

9 ATG AGT TTA GTT AAT AGA AAA CAG TTA GAA AAA ATG GCA AAT GTA AGA 48 Met Ser Leu Val Asn Arg Lys Gln Leu Glu Lys Met Ala Asn Val Arg 1 5 10 15 TTT CGT ACT CAA GAA GAT GAA TAT GTT GCA ATA TTG GAT GCT TTA GAA 96 Phe Arg Thr Gln Glu Asp Glu Tyr Val Ala Ile Leu Asp Ala Leu Glu 20 25 30 GAA TAT CAT AAT ATG TCA GAG AAT ACT GTA GTC GAA AAA TAT TTA AAA 144 Glu Tyr His Asn Met Ser Glu Asn Thr Val Val Glu Lys Tyr Leu Lys 35 40 45 TTA AAA GAT ATA AAT AGT TTA ACA GAT ATT TAT ATA GAT ACA TAT AAA 192 Leu Lys Asp Ile Asn Ser Leu Thr Asp Ile Tyr Ile Asp Thr Tyr Lys 50 55 60 AAA TCT GGT AGA AAT AAA GCC TTA AAA AAA TTT AAG GAA TAT CTA GTT 240 Lys Ser Gly Arg Asn Lys Ala Leu Lys Lys Phe Lys Glu Tyr Leu Val 65 70 75 80 ACA GAA GTA TTA GAG CTA AAG AAT AAT AAT TTA ACT CCA GTT GAG AAA 288 Thr Glu Val Leu Glu Leu Lys Asn Asn Asn Leu Thr Pro Val Glu Lys 85 90 95 AAT TTA CAT TTT GTT TGG ATT GGA GGT CAA ATA AAT GAC ACT GCT ATT 336 Asn Leu His Phe Val Trp Ile Gly Gly Gln Ile Asn Asp Thr Ala Ile 100 105 110 AAT TAT ATA AAT CAA TGG AAA GAT GTA AAT AGT GAT TAT AAT GTT AAT 384 Asn Tyr Ile Asn Gln Trp Lys Asp Val Asn Ser Asp Tyr Asn Val Asn 115 120 125 GTT TTT TAT GAT AGT AAT GCA TTT TTG ATA AAC ACA TTG AAA AAA ACT 432 Val Phe Tyr Asp Ser Asn Ala Phe Leu Ile Asn Thr Leu Lys Lys Thr 130 135 140 GTA GTA GAA TCA GCA ATA AAT GAT ACA CTT GAA TCA TTT AGA GAA AAC 480 Val Val Glu Ser Ala Ile Asn Asp Thr Leu Glu Ser Phe Arg Glu Asn 145 150 155 160 TTA AAT GAC CCT AGA TTT GAC TAT AAT AAA TTC TTC AGA AAA CGT ATG 528 Leu Asn Asp Pro Arg Phe Asp Tyr Asn Lys Phe Phe Arg Lys Arg Met 165 170 175 GAA ATA ATT TAT GAT AAA CAG AAA AAT TTC ATA AAC TAC TAT AAA GCT 576 Glu Ile Ile Tyr Asp Lys Gln Lys Asn Phe Ile Asn Tyr Tyr Lys Ala 180 185 190 CAA AGA GAA GAA AAT CCT GAA CTT ATA ATT GAT GAT ATT GTA AAG ACA 624 Gln Arg Glu Glu Asn Pro Glu Leu Ile Ile Asp Asp Ile Val Lys Thr 195 200 205 TAT CTT TCA AAT GAG TAT TCA AAG GAG ATA GAT GAA CTT AAT ACC TAT 672 Tyr Leu Ser Asn Glu Tyr Ser Lys Glu Ile Asp Glu Leu Asn Thr Tyr 210 215 220 ATT GAA GAA TCC TTA AAT AAA ATT ACA CAG AAT AGT GGA AAT GAT GTT 720 Ile Glu Glu Ser Leu Asn Lys Ile Thr Gln Asn Ser Gly Asn Asp Val 225 230 235 240 AGA AAC TTT GAA GAA TTT AAA AAT GGA GAG TCA TTC AAC TTA TAT GAA 768 Arg Asn Phe Glu Glu Phe Lys Asn Gly Glu Ser Phe Asn Leu Tyr Glu 245 250 255 CAA GAG TTG GTA GAA AGG TGG AAT TTA GCT GCT GCT TCT GAC ATA TTA 816 Gln Glu Leu Val Glu Arg Trp Asn Leu Ala Ala Ala Ser Asp Ile Leu 260 265 270 AGA ATA TCT GCA TTA AAA GAA ATT GGT GGT ATG TAT TTA GAT GTT GAT 864 Arg Ile Ser Ala Leu Lys Glu Ile Gly Gly Met Tyr Leu Asp Val Asp 275 280 285 ATG TTA CCA GGA ATA CAA CCA GAC TTA TTT GAG TCT ATA GAG AAA CCT 912 Met Leu Pro Gly Ile Gln Pro Asp Leu Phe Glu Ser Ile Glu Lys Pro 290 295 300 AGT TCA GTA ACA GTG GAT TTT TGG GAA ATG ACA AAG TTA GAA GCT ATA 960 Ser Ser Val Thr Val Asp Phe Trp Glu Met Thr Lys Leu Glu Ala Ile 305 310 315 320 ATG AAA TAC AAA GAA TAT ATA CCA GAA TAT ACC TCA GAA CAT TTT GAC 1008 Met Lys Tyr Lys Glu Tyr Ile Pro Glu Tyr Thr Ser Glu His Phe Asp 325 330 335 ATG TTA GAC GAA GAA GTT CAA AGT AGT TTT GAA TCT GTT CTA GCT TCT 1056 Met Leu Asp Glu Glu Val Gln Ser Ser Phe Glu Ser Val Leu Ala Ser 340 345 350 AAG TCA GAT AAA TCA GAA ATA TTC TCA TCA CTT GGT GAT ATG GAG GCA 1104 Lys Ser Asp Lys Ser Glu Ile Phe Ser Ser Leu Gly Asp Met Glu Ala 355 360 365 TCA CCA CTA GAA GTT AAA ATT GCA TTT AAT AGT AAG GGT ATT ATA AAT 1152 Ser Pro Leu Glu Val Lys Ile Ala Phe Asn Ser Lys Gly Ile Ile Asn 370 375 380 CAA GGG CTA ATT TCT GTG AAA GAC TCA TAT TGT AGC AAT TTA ATA GTA 1200 Gln Gly Leu Ile Ser Val Lys Asp Ser Tyr Cys Ser Asn Leu Ile Val 385 390 395 400 AAA CAA ATC GAG AAT AGA TAT AAA ATA TTG AAT AAT AGT TTA AAT CCA 1248 Lys Gln Ile Glu Asn Arg Tyr Lys Ile Leu Asn Asn Ser Leu Asn Pro 405 410 415 GCT ATT AGC GAG GAT AAT GAT TTT AAT ACT ACA ACG AAT ACC TTT ATT 1296 Ala Ile Ser Glu Asp Asn Asp Phe Asn Thr Thr Thr Asn Thr Phe Ile 420 425 430 GAT AGT ATA ATG GCT GAA GCT AAT GCA GAT AAT GGT AGA TTT ATG ATG 1344 Asp Ser Ile Met Ala Glu Ala Asn Ala Asp Asn Gly Arg Phe Met Met 435 440 445 GAA CTA GGA AAG TAT TTA AGA GTT GGT TTC TTC CCA GAT GTT AAA ACT 1392 Glu Leu Gly Lys Tyr Leu Arg Val Gly Phe Phe Pro Asp Val Lys Thr 450 455 460 ACT ATT AAC TTA AGT GGC CCT GAA GCA TAT GCG GCA GCT TAT CAA GAT 1440 Thr Ile Asn Leu Ser Gly Pro Glu Ala Tyr Ala Ala Ala Tyr Gln Asp 465 470 475 480 TTA TTA ATG TTT AAA GAA GGC AGT ATG AAT ATC CAT TTG ATA GAA GCT 1488 Leu Leu Met Phe Lys Glu Gly Ser Met Asn Ile His Leu Ile Glu Ala 485 490 495 GAT TTA AGA AAC TTT GAA ATC TCT AAA ACT AAT ATT TCT CAA TCA ACT 1536 Asp Leu Arg Asn Phe Glu Ile Ser Lys Thr Asn Ile Ser Gln Ser Thr 500 505 510 GAA CAA GAA ATG GCT AGC TTA TGG TCA TTT GAC GAT GCA AGA GCT AAA 1584 Glu Gln Glu Met Ala Ser Leu Trp Ser Phe Asp Asp Ala Arg Ala Lys 515 520 525 GCT CAA TTT GAA GAA TAT AAA AGG AAT TAT TTT GAA GGT TCT CTT GGT 1632 Ala Gln Phe Glu Glu Tyr Lys Arg Asn Tyr Phe Glu Gly Ser Leu Gly 530 535 540 GAA GAT GAT AAT CTT GAT TTT TCT CAA AAT ATA GTA GTT GAC AAG GAG 1680 Glu Asp Asp Asn Leu Asp Phe Ser Gln Asn Ile Val Val Asp Lys Glu 545 550 555 560 TAT CTT TTA GAA AAA ATA TCT TCA TTA GCA AGA AGT TCA GAG AGA GGA 1728 Tyr Leu Leu Glu Lys Ile Ser Ser Leu Ala Arg Ser Ser Glu Arg Gly 565 570 575 TAT ATA CAC TAT ATT GTT CAG TTA CAA GGA GAT AAA ATT AGT TAT GAA 1776 Tyr Ile His Tyr Ile Val Gln Leu Gln Gly Asp Lys Ile Ser Tyr Glu 580 585 590 GCA GCA TGT AAC TTA TTT GCA AAG ACT CCT TAT GAT AGT GTA CTG TTT 1824 Ala Ala Cys Asn Leu Phe Ala Lys Thr Pro Tyr Asp Ser Val Leu Phe 595 600 605 CAG AAA AAT ATA GAA GAT TCA GAA ATT GCA TAT TAT TAT AAT CCT GGA 1872 Gln Lys Asn Ile Glu Asp Ser Glu Ile Ala Tyr Tyr Tyr Asn Pro Gly 610 615 620 GAT GGT GAA ATA CAA GAA ATA GAC AAG TAT AAA ATT CCA AGT ATA ATT 1920 Asp Gly Glu Ile Gln Glu Ile Asp Lys Tyr Lys Ile Pro Ser Ile Ile 625 630 635 640 TCT GAT AGA CCT AAG ATT AAA TTA ACA TTT ATT GGT CAT GGT AAA GAT 1968 Ser Asp Arg Pro Lys Ile Lys Leu Thr Phe Ile Gly His Gly Lys Asp 645 650 655 GAA TTT AAT ACT GAT ATA TTT GCA GGT TTT GAT GTA GAT TCA TTA TCC 2016 Glu Phe Asn Thr Asp Ile Phe Ala Gly Phe Asp Val Asp Ser Leu Ser 660 665 670 ACA GAA ATA GAA GCA GCA ATA GAT TTA GCT AAA GAG GAT ATT TCT CCT 2064 Thr Glu Ile Glu Ala Ala Ile Asp Leu Ala Lys Glu Asp Ile Ser Pro 675 680 685 AAG TCA ATA GAA ATA AAT TTA TTA GGA TGT AAT ATG TTT AGC TAC TCT 2112 Lys Ser Ile Glu Ile Asn Leu Leu Gly Cys Asn Met Phe Ser Tyr Ser 690 695 700 ATC AAC GTA GAG GAG ACT TAT CCT GGA AAA TTA TTA CTT AAA GTT AAA 2160 Ile Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu Leu Leu Lys Val Lys 705 710 715 720 GAT AAA ATA TCA GAA TTA ATG CCA TCT ATA AGT CAA GAC TCT ATT ATA 2208 Asp Lys Ile Ser Glu Leu Met Pro Ser Ile Ser Gln Asp Ser Ile Ile 725 730 735 GTA AGT GCA AAT CAA TAT GAA GTT AGA ATA AAT AGT GAA GGA AGA AGA 2256 Val Ser Ala Asn Gln Tyr Glu Val Arg Ile Asn Ser Glu Gly Arg Arg 740 745 750 GAA TTA TTG GAT CAT TCT GGT GAA TGG ATA AAT AAA GAA GAA AGT ATT 2304 Glu Leu Leu Asp His Ser Gly Glu Trp Ile Asn Lys Glu Glu Ser Ile 755 760 765 ATA AAG GAT ATT TCA TCA AAA GAA TAT ATA TCA TTT AAT CCT AAA GAA 2352 Ile Lys Asp Ile Ser Ser Lys Glu Tyr Ile Ser Phe Asn Pro Lys Glu 770 775 780 AAT AAA ATT ACA GTA AAA TCT AAA AAT TTA CCT GAG CTA TCT ACA TTA 2400 Asn Lys Ile Thr Val Lys Ser Lys Asn Leu Pro Glu Leu Ser Thr Leu 785 790 795 800 TTA CAA GAA ATT AGA AAT AAT TCT AAT TCA AGT GAT ATT GAA CTA GAA 2448 Leu Gln Glu Ile Arg Asn Asn Ser Asn Ser Ser Asp Ile Glu Leu Glu 805 810 815 GAA AAA GTA ATG TTA ACA GAA TGT GAG ATA AAT GTT ATT TCA AAT ATA 2496 Glu Lys Val Met Leu Thr Glu Cys Glu Ile Asn Val Ile Ser Asn Ile 820 825 830 GAT ACG CAA ATT GTT GAG GAA AGG ATT GAA GAA GCT AAG AAT TTA ACT 2544 Asp Thr Gln Ile Val Glu Glu Arg Ile Glu Glu Ala Lys Asn Leu Thr 835 840 845 TCT GAC TCT ATT AAT TAT ATA AAA GAT GAA TTT AAA CTA ATA GAA TCT 2592 Ser Asp Ser Ile Asn Tyr Ile Lys Asp Glu Phe Lys Leu Ile Glu Ser 850 855 860 ATT TCT GAT GCA CTA TGT GAC TTA AAA CAA CAG AAT GAA TTA GAA GAT 2640 Ile Ser Asp Ala Leu Cys Asp Leu Lys Gln Gln Asn Glu Leu Glu Asp 865 870 875 880 TCT CAT TTT ATA TCT TTT GAG GAC ATA TCA GAG ACT GAT GAG GGA TTT 2688 Ser His Phe Ile Ser Phe Glu Asp Ile Ser Glu Thr Asp Glu Gly Phe 885 890 895 AGT ATA AGA TTT ATT AAT AAA GAA ACT GGA GAA TCT ATA TTT GTA GAA 2736 Ser Ile Arg Phe Ile Asn Lys Glu Thr Gly Glu Ser Ile Phe Val Glu 900 905 910 ACT GAA AAA ACA ATA TTC TCT GAA TAT GCT AAT CAT ATA ACT GAA GAG 2784 Thr Glu Lys Thr Ile Phe Ser Glu Tyr Ala Asn His Ile Thr Glu Glu 915 920 925 ATT TCT AAG ATA AAA GGT ACT ATA TTT GAT ACT GTA AAT GGT AAG TTA 2832 Ile Ser Lys Ile Lys Gly Thr Ile Phe Asp Thr Val Asn Gly Lys Leu 930 935 940 GTA AAA AAA GTA AAT TTA GAT ACT ACA CAC GAA GTA AAT ACT TTA AAT 2880 Val Lys Lys Val Asn Leu Asp Thr Thr His Glu Val Asn Thr Leu Asn 945 950 955 960 GCT GCA TTT TTT ATA CAA TCA TTA ATA GAA TAT AAT AGT TCT AAA GAA 2928 Ala Ala Phe Phe Ile Gln Ser Leu Ile Glu Tyr Asn Ser Ser Lys Glu 965 970 975 TCT CTT AGT AAT TTA AGT GTA GCA ATG AAA GTC CAA GTT TAC GCT CAA 2976 Ser Leu Ser Asn Leu Ser Val Ala Met Lys Val Gln Val Tyr Ala Gln 980 985 990 TTA TTT AGT ACT GGT TTA AAT ACT ATT ACA GAT GCA GCC AAA GTT GTT 3024 Leu Phe Ser Thr Gly Leu Asn Thr Ile Thr Asp Ala Ala Lys Val Val 995 1000 1005 GAA TTA GTA TCA ACT GCA TTA GAT GAA ACT ATA GAC TTA CTT CCT ACA 3072 Glu Leu Val Ser Thr Ala Leu Asp Glu Thr Ile Asp Leu Leu Pro Thr 1010 1015 1020 TTA TCT GAA GGA TTA CCT ATA ATT GCA ACT ATT ATA GAT GGT GTA AGT 3120 Leu Ser Glu Gly Leu Pro Ile Ile Ala Thr Ile Ile Asp Gly Val Ser 1025 1030 1035 1040 TTA GGT GCA GCA ATC AAA GAG CTA AGT GAA ACG AGT GAC CCA TTA TTA 3168 Leu Gly Ala Ala Ile Lys Glu Leu Ser Glu Thr Ser Asp Pro Leu Leu 1045 1050 1055 AGA CAA GAA ATA GAA GCT AAG ATA GGT ATA ATG GCA GTA AAT TTA ACA 3216 Arg Gln Glu Ile Glu Ala Lys Ile Gly Ile Met Ala Val Asn Leu Thr 1060 1065 1070 ACA GCT ACA ACT GCA ATC ATT ACT TCA TCT TTG GGG ATA GCT AGT GGA 3264 Thr Ala Thr Thr Ala Ile Ile Thr Ser Ser Leu Gly Ile Ala Ser Gly 1075 1080 1085 TTT AGT ATA CTT TTA GTT CCT TTA GCA GGA ATT TCA GCA GGT ATA CCA 3312 Phe Ser Ile Leu Leu Val Pro Leu Ala Gly Ile Ser Ala Gly Ile Pro 1090 1095 1100 AGC TTA GTA AAC AAT GAA CTT GTA CTT CGA GAT AAG GCA ACA AAG GTT 3360 Ser Leu Val Asn Asn Glu Leu Val Leu Arg Asp Lys Ala Thr Lys Val 1105 1110 1115 1120 GTA GAT TAT TTT AAA CAT GTT TCA TTA GTT GAA ACT GAA GGA GTA TTT 3408 Val Asp Tyr Phe Lys His Val Ser Leu Val Glu Thr Glu Gly Val Phe 1125 1130 1135 ACT TTA TTA GAT GAT AAA ATA ATG ATG CCA CAA GAT GAT TTA GTG ATA 3456 Thr Leu Leu Asp Asp Lys Ile Met Met Pro Gln Asp Asp Leu Val Ile 1140 1145 1150 TCA GAA ATA GAT TTT AAT AAT AAT TCA ATA GTT TTA GGT AAA TGT GAA 3504 Ser Glu Ile Asp Phe Asn Asn Asn Ser Ile Val Leu Gly Lys Cys Glu 1155 1160 1165 ATC TGG AGA ATG GAA GGT GGT TCA GGT CAT ACT GTA ACT GAT GAT ATA 3552 Ile Trp Arg Met Glu Gly Gly Ser Gly His Thr Val Thr Asp Asp Ile 1170 1175 1180 GAT CAC TTC TTT TCA GCA CCA TCA ATA ACA TAT AGA GAG CCA CAC TTA 3600 Asp His Phe Phe Ser Ala Pro Ser Ile Thr Tyr Arg Glu Pro His Leu 1185 1190 1195 1200 TCT ATA TAT GAC GTA TTG GAA GTA CAA AAA GAA GAA CTT GAT TTG TCA 3648 Ser Ile Tyr Asp Val Leu Glu Val Gln Lys Glu Glu Leu Asp Leu Ser 1205 1210 1215 AAA GAT TTA ATG GTA TTA CCT AAT GCT CCA AAT AGA GTA TTT GCT TGG 3696 Lys Asp Leu Met Val Leu Pro Asn Ala Pro Asn Arg Val Phe Ala Trp 1220 1225 1230 GAA ACA GGA TGG ACA CCA GGT TTA AGA AGC TTA GAA AAT GAT GGC ACA 3744 Glu Thr Gly Trp Thr Pro Gly Leu Arg Ser Leu Glu Asn Asp Gly Thr 1235 1240 1245 AAA CTG TTA GAC CGT ATA AGA GAT AAC TAT GAA GGT GAG TTT TAT TGG 3792 Lys Leu Leu Asp Arg Ile Arg Asp Asn Tyr Glu Gly Glu Phe Tyr Trp 1250 1255 1260 AGA TAT TTT GCT TTT ATA GCT GAT GCT TTA ATA ACA ACA TTA AAA CCA 3840 Arg Tyr Phe Ala Phe Ile Ala Asp Ala Leu Ile Thr Thr Leu Lys Pro 1265 1270 1275 1280 AGA TAT GAA GAT ACT AAT ATA AGA ATA AAT TTA GAT AGT AAT ACT AGA 3888 Arg Tyr Glu Asp Thr Asn Ile Arg Ile Asn Leu Asp Ser Asn Thr Arg 1285 1290 1295 AGT TTT ATA GTT CCA ATA ATA ACT ACA GAA TAT ATA AGA GAA AAA TTA 3936 Ser Phe Ile Val Pro Ile Ile Thr Thr Glu Tyr Ile Arg Glu Lys Leu 1300 1305 1310 TCA TAT TCT TTC TAT GGT TCA GGA GGA ACT TAT GCA TTG TCT CTT TCT 3984 Ser Tyr Ser Phe Tyr Gly Ser Gly Gly Thr Tyr Ala Leu Ser Leu Ser 1315 1320 1325 CAA TAT AAT ATG GGT ATA AAT ATA GAA TTA AGT GAA AGT GAT GTT TGG 4032 Gln Tyr Asn Met Gly Ile Asn Ile Glu Leu Ser Glu Ser Asp Val Trp 1330 1335 1340 ATT ATA GAT GTT GAT AAT GTT GTG AGA GAT GTA ACT ATA GAA TCT GAT 4080 Ile Ile Asp Val Asp Asn Val Val Arg Asp Val Thr Ile Glu Ser Asp 1345 1350 1355 1360 AAA ATT AAA AAA GGT GAT TTA ATA GAA GGT ATT TTA TCT ACA CTA AGT 4128 Lys Ile Lys Lys Gly Asp Leu Ile Glu Gly Ile Leu Ser Thr Leu Ser 1365 1370 1375 ATT GAA GAG AAT AAA ATT ATC TTA AAT AGC CAT GAG ATT AAT TTT TCT 4176 Ile Glu Glu Asn Lys Ile Ile Leu Asn Ser His Glu Ile Asn Phe Ser 1380 1385 1390 GGT GAG GTA AAT GGA AGT AAT GGA TTT GTT TCT TTA ACA TTT TCA ATT 4224 Gly Glu Val Asn Gly Ser Asn Gly Phe Val Ser Leu Thr Phe Ser Ile 1395 1400 1405 TTA GAA GGA ATA AAT GCA ATT ATA GAA GTT GAT TTA TTA TCT AAA TCA 4272 Leu Glu Gly Ile Asn Ala Ile Ile Glu Val Asp Leu Leu Ser Lys Ser 1410 1415 1420 TAT AAA TTA CTT ATT TCT GGC GAA TTA AAA ATA TTG ATG TTA AAT TCA 4320 Tyr Lys Leu Leu Ile Ser Gly Glu Leu Lys Ile Leu Met Leu Asn Ser 1425 1430 1435 1440 AAT CAT ATT CAA CAG AAA ATA GAT TAT ATA GGA TTC AAT AGC GAA TTA 4368 Asn His Ile Gln Gln Lys Ile Asp Tyr Ile Gly Phe Asn Ser Glu Leu 1445 1450 1455 CAG AAA AAT ATA CCA TAT AGC TTT GTA GAT AGT GAA GGA AAA GAG AAT 4416 Gln Lys Asn Ile Pro Tyr Ser Phe Val Asp Ser Glu Gly Lys Glu Asn 1460 1465 1470 GGT TTT ATT AAT GGT TCA ACA AAA GAA GGT TTA TTT GTA TCT GAA TTA 4464 Gly Phe Ile Asn Gly Ser Thr Lys Glu Gly Leu Phe Val Ser Glu Leu 1475 1480 1485 CCT GAT GTA GTT CTT ATA AGT AAG GTT TAT ATG GAT GAT AGT AAG CCT 4512 Pro Asp Val Val Leu Ile Ser Lys Val Tyr Met Asp Asp Ser Lys Pro 1490 1495 1500 TCA TTT GGA TAT TAT AGT AAT AAT TTG AAA GAT GTC AAA GTT ATA ACT 4560 Ser Phe Gly Tyr Tyr Ser Asn Asn Leu Lys Asp Val Lys Val Ile Thr 1505 1510 1515 1520 AAA GAT AAT GTT AAT ATA TTA ACA GGT TAT TAT CTT AAG GAT GAT ATA 4608 Lys Asp Asn Val Asn Ile Leu Thr Gly Tyr Tyr Leu Lys Asp Asp Ile 1525 1530 1535 AAA ATC TCT CTT TCT TTG ACT CTA CAA GAT GAA AAA ACT ATA AAG TTA 4656 Lys Ile Ser Leu Ser Leu Thr Leu Gln Asp Glu Lys Thr Ile Lys Leu 1540 1545 1550 AAT AGT GTG CAT TTA GAT GAA AGT GGA GTA GCT GAG ATT TTG AAG TTC 4704 Asn Ser Val His Leu Asp Glu Ser Gly Val Ala Glu Ile Leu Lys Phe 1555 1560 1565 ATG AAT AGA AAA GGT AAT ACA AAT ACT TCA GAT TCT TTA ATG AGC TTT 4752 Met Asn Arg Lys Gly Asn Thr Asn Thr Ser Asp Ser Leu Met Ser Phe 1570 1575 1580 TTA GAA AGT ATG AAT ATA AAA AGT ATT TTC GTT AAT TTC TTA CAA TCT 4800 Leu Glu Ser Met Asn Ile Lys Ser Ile Phe Val Asn Phe Leu Gln Ser 1585 1590 1595 1600 AAT ATT AAG TTT ATA TTA GAT GCT AAT TTT ATA ATA AGT GGT ACT ACT 4848 Asn Ile Lys Phe Ile Leu Asp Ala Asn Phe Ile Ile Ser Gly Thr Thr 1605 1610 1615 TCT ATT GGC CAA TTT GAG TTT ATT TGT GAT GAA AAT GAT AAT ATA CAA 4896 Ser Ile Gly Gln Phe Glu Phe Ile Cys Asp Glu Asn Asp Asn Ile Gln 1620 1625 1630 CCA TAT TTC ATT AAG TTT AAT ACA CTA GAA ACT AAT TAT ACT TTA TAT 4944 Pro Tyr Phe Ile Lys Phe Asn Thr Leu Glu Thr Asn Tyr Thr Leu Tyr 1635 1640 1645 GTA GGA AAT AGA CAA AAT ATG ATA GTG GAA CCA AAT TAT GAT TTA GAT 4992 Val Gly Asn Arg Gln Asn Met Ile Val Glu Pro Asn Tyr Asp Leu Asp 1650 1655 1660 GAT TCT GGA GAT ATA TCT TCA ACT GTT ATC AAT TTC TCT CAA AAG TAT 5040 Asp Ser Gly Asp Ile Ser Ser Thr Val Ile Asn Phe Ser Gln Lys Tyr 1665 1670 1675 1680 CTT TAT GGA ATA GAC AGT TGT GTT AAT AAA GTT GTA ATT TCA CCA AAT 5088 Leu Tyr Gly Ile Asp Ser Cys Val Asn Lys Val Val Ile Ser Pro Asn 1685 1690 1695 ATT TAT ACA GAT GAA ATA AAT ATA ACG CCT GTA TAT GAA ACA AAT AAT 5136 Ile Tyr Thr Asp Glu Ile Asn Ile Thr Pro Val Tyr Glu Thr Asn Asn 1700 1705 1710 ACT TAT CCA GAA GTT ATT GTA TTA GAT GCA AAT TAT ATA AAT GAA AAA 5184 Thr Tyr Pro Glu Val Ile Val Leu Asp Ala Asn Tyr Ile Asn Glu Lys 1715 1720 1725 ATA AAT GTT AAT ATC AAT GAT CTA TCT ATA CGA TAT GTA TGG AGT AAT 5232 Ile Asn Val Asn Ile Asn Asp Leu Ser Ile Arg Tyr Val Trp Ser Asn 1730 1735 1740 GAT GGT AAT GAT TTT ATT CTT ATG TCA ACT AGT GAA GAA AAT AAG GTG 5280 Asp Gly Asn Asp Phe Ile Leu Met Ser Thr Ser Glu Glu Asn Lys Val 1745 1750 1755 1760 TCA CAA GTT AAA ATA AGA TTC GTT AAT GTT TTT AAA GAT AAG ACT TTG 5328 Ser Gln Val Lys Ile Arg Phe Val Asn Val Phe Lys Asp Lys Thr Leu 1765 1770 1775 GCA AAT AAG CTA TCT TTT AAC TTT AGT GAT AAA CAA GAT GTA CCT GTA 5376 Ala Asn Lys Leu Ser Phe Asn Phe Ser Asp Lys Gln Asp Val Pro Val 1780 1785 1790 AGT GAA ATA ATC TTA TCA TTT ACA CCT TCA TAT TAT GAG GAT GGA TTG 5424 Ser Glu Ile Ile Leu Ser Phe Thr Pro Ser Tyr Tyr Glu Asp Gly Leu 1795 1800 1805 ATT GGC TAT GAT TTG GGT CTA GTT TCT TTA TAT AAT GAG AAA TTT TAT 5472 Ile Gly Tyr Asp Leu Gly Leu Val Ser Leu Tyr Asn Glu Lys Phe Tyr 1810 1815 1820 ATT AAT AAC TTT GGA ATG ATG GTA TCT GGA TTA ATA TAT ATT AAT GAT 5520 Ile Asn Asn Phe Gly Met Met Val Ser Gly Leu Ile Tyr Ile Asn Asp 1825 1830 1835 1840 TCA TTA TAT TAT TTT AAA CCA CCA GTA AAT AAT TTG ATA ACT GGA TTT 5568 Ser Leu Tyr Tyr Phe Lys Pro Pro Val Asn Asn Leu Ile Thr Gly Phe 1845 1850 1855 GTG ACT GTA GGC GAT GAT AAA TAC TAC TTT AAT CCA ATT AAT GGT GGA 5616 Val Thr Val Gly Asp Asp Lys Tyr Tyr Phe Asn Pro Ile Asn Gly Gly 1860 1865 1870 GCT GCT TCA ATT GGA GAG ACA ATA ATT GAT GAC AAA AAT TAT TAT TTC 5664 Ala Ala Ser Ile Gly Glu Thr Ile Ile Asp Asp Lys Asn Tyr Tyr Phe 1875 1880 1885 AAC CAA AGT GGA GTG TTA CAA ACA GGT GTA TTT AGT ACA GAA GAT GGA 5712 Asn Gln Ser Gly Val Leu Gln Thr Gly Val Phe Ser Thr Glu Asp Gly 1890 1895 1900 TTT AAA TAT TTT GCC CCA GCT AAT ACA CTT GAT GAA AAC CTA GAA GGA 5760 Phe Lys Tyr Phe Ala Pro Ala Asn Thr Leu Asp Glu Asn Leu Glu Gly 1905 1910 1915 1920 GAA GCA ATT GAT TTT ACT GGA AAA TTA ATT ATT GAC GAA AAT ATT TAT 5808 Glu Ala Ile Asp Phe Thr Gly Lys Leu Ile Ile Asp Glu Asn Ile Tyr 1925 1930 1935 TAT TTT GAT GAT AAT TAT AGA GGA GCT GTA GAA TGG AAA GAA TTA GAT 5856 Tyr Phe Asp Asp Asn Tyr Arg Gly Ala Val Glu Trp Lys Glu Leu Asp 1940 1945 1950 GGT GAA ATG CAC TAT TTT AGC CCA GAA ACA GGT AAA GCT TTT AAA GGT 5904 Gly Glu Met His Tyr Phe Ser Pro Glu Thr Gly Lys Ala Phe Lys Gly 1955 1960 1965 CTA AAT CAA ATA GGT GAT TAT AAA TAC TAT TTC AAT TCT GAT GGA GTT 5952 Leu Asn Gln Ile Gly Asp Tyr Lys Tyr Tyr Phe Asn Ser Asp Gly Val 1970 1975 1980 ATG CAA AAA GGA TTT GTT AGT ATA AAT GAT AAT AAA CAC TAT TTT GAT 6000 Met Gln Lys Gly Phe Val Ser Ile Asn Asp Asn Lys His Tyr Phe Asp 1985 1990 1995 2000 GAT TCT GGT GTT ATG AAA GTA GGT TAC ACT GAA ATA GAT GGC AAG CAT 6048 Asp Ser Gly Val Met Lys Val Gly Tyr Thr Glu Ile Asp Gly Lys His 2005 2010 2015 TTC TAC TTT GCT GAA AAC GGA GAA ATG CAA ATA GGA GTA TTT AAT ACA 6096 Phe Tyr Phe Ala Glu Asn Gly Glu Met Gln Ile Gly Val Phe Asn Thr 2020 2025 2030 GAA GAT GGA TTT AAA TAT TTT GCT CAT CAT AAT GAA GAT TTA GGA AAT 6144 Glu Asp Gly Phe Lys Tyr Phe Ala His His Asn Glu Asp Leu Gly Asn 2035 2040 2045 GAA GAA GGT GAA GAA ATC TCA TAT TCT GGT ATA TTA AAT TTC AAT AAT 6192 Glu Glu Gly Glu Glu Ile Ser Tyr Ser Gly Ile Leu Asn Phe Asn Asn 2050 2055 2060 AAA ATT TAC TAT TTT GAT GAT TCA TTT ACA GCT GTA GTT GGA TGG AAA 6240 Lys Ile Tyr Tyr Phe Asp Asp Ser Phe Thr Ala Val Val Gly Trp Lys 2065 2070 2075 2080 GAT TTA GAG GAT GGT TCA AAG TAT TAT TTT GAT GAA GAT ACA GCA GAA 6288 Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe Asp Glu Asp Thr Ala Glu 2085 2090 2095 GCA TAT ATA GGT TTG TCA TTA ATA AAT GAT GGT CAA TAT TAT TTT AAT 6336 Ala Tyr Ile Gly Leu Ser Leu Ile Asn Asp Gly Gln Tyr Tyr Phe Asn 2100 2105 2110 GAT GAT GGA ATT ATG CAA GTT GGA TTT GTC ACT ATA AAT GAT AAA GTC 6384 Asp Asp Gly Ile Met Gln Val Gly Phe Val Thr Ile Asn Asp Lys Val 2115 2120 2125 TTC TAC TTC TCT GAC TCT GGA ATT ATA GAA TCT GGA GTA CAA AAC ATA 6432 Phe Tyr Phe Ser Asp Ser Gly Ile Ile Glu Ser Gly Val Gln Asn Ile 2130 2135 2140 GAT GAC AAT TAT TTC TAT ATA GAT GAT AAT GGT ATA GTT CAA ATT GGT 6480 Asp Asp Asn Tyr Phe Tyr Ile Asp Asp Asn Gly Ile Val Gln Ile Gly 2145 2150 2155 2160 GTA TTT GAT ACT TCA GAT GGA TAT AAA TAT TTT GCA CCT GCT AAT ACT 6528 Val Phe Asp Thr Ser Asp Gly Tyr Lys Tyr Phe Ala Pro Ala Asn Thr 2165 2170 2175 GTA AAT GAT AAT ATT TAC GGA CAA GCA GTT GAA TAT AGT GGT TTA GTT 6576 Val Asn Asp Asn Ile Tyr Gly Gln Ala Val Glu Tyr Ser Gly Leu Val 2180 2185 2190 AGA GTT GGG GAA GAT GTA TAT TAT TTT GGA GAA ACA TAT ACA ATT GAG 6624 Arg Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Thr Ile Glu 2195 2200 2205 ACT GGA TGG ATA TAT GAT ATG GAA AAT GAA AGT GAT AAA TAT TAT TTC 6672 Thr Gly Trp Ile Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr Tyr Phe 2210 2215 2220 AAT CCA GAA ACT AAA AAA GCA TGC AAA GGT ATT AAT TTA ATT GAT GAT 6720 Asn Pro Glu Thr Lys Lys Ala Cys Lys Gly Ile Asn Leu Ile Asp Asp 2225 2230 2235 2240 ATA AAA TAT TAT TTT GAT GAG AAG GGC ATA ATG AGA ACG GGT CTT ATA 6768 Ile Lys Tyr Tyr Phe Asp Glu Lys Gly Ile Met Arg Thr Gly Leu Ile 2245 2250 2255 TCA TTT GAA AAT AAT AAT TAT TAC TTT AAT GAG AAT GGT GAA ATG CAA 6816 Ser Phe Glu Asn Asn Asn Tyr Tyr Phe Asn Glu Asn Gly Glu Met Gln 2260 2265 2270 TTT GGT TAT ATA AAT ATA GAA GAT AAG ATG TTC TAT TTT GGT GAA GAT 6864 Phe Gly Tyr Ile Asn Ile Glu Asp Lys Met Phe Tyr Phe Gly Glu Asp 2275 2280 2285 GGT GTC ATG CAG ATT GGA GTA TTT AAT ACA CCA GAT GGA TTT AAA TAC 6912 Gly Val Met Gln Ile Gly Val Phe Asn Thr Pro Asp Gly Phe Lys Tyr 2290 2295 2300 TTT GCA CAT CAA AAT ACT TTG GAT GAG AAT TTT GAG GGA GAA TCA ATA 6960 Phe Ala His Gln Asn Thr Leu Asp Glu Asn Phe Glu Gly Glu Ser Ile 2305 2310 2315 2320 AAC TAT ACT GGT TGG TTA GAT TTA GAT GAA AAG AGA TAT TAT TTT ACA 7008 Asn Tyr Thr Gly Trp Leu Asp Leu Asp Glu Lys Arg Tyr Tyr Phe Thr 2325 2330 2335 GAT GAA TAT ATT GCA GCA ACT GGT TCA GTT ATT ATT GAT GGT GAG GAG 7056 Asp Glu Tyr Ile Ala Ala Thr Gly Ser Val Ile Ile Asp Gly Glu Glu 2340 2345 2350 TAT TAT TTT GAT CCT GAT ACA GCT CAA TTA GTG ATT AGT GAA 7098 Tyr Tyr Phe Asp Pro Asp Thr Ala Gln Leu Val Ile Ser Glu 2355 2360 2365 TAG 7101

10 Met Ser Leu Val Asn Arg Lys Gln Leu Glu Lys Met Ala Asn Val Arg 1 5 10 15 Phe Arg Thr Gln Glu Asp Glu Tyr Val Ala Ile Leu Asp Ala Leu Glu 20 25 30 Glu Tyr His Asn Met Ser Glu Asn Thr Val Val Glu Lys Tyr Leu Lys 35 40 45 Leu Lys Asp Ile Asn Ser Leu Thr Asp Ile Tyr Ile Asp Thr Tyr Lys 50 55 60 Lys Ser Gly Arg Asn Lys Ala Leu Lys Lys Phe Lys Glu Tyr Leu Val 65 70 75 80 Thr Glu Val Leu Glu Leu Lys Asn Asn Asn Leu Thr Pro Val Glu Lys 85 90 95 Asn Leu His Phe Val Trp Ile Gly Gly Gln Ile Asn Asp Thr Ala Ile 100 105 110 Asn Tyr Ile Asn Gln Trp Lys Asp Val Asn Ser Asp Tyr Asn Val Asn 115 120 125 Val Phe Tyr Asp Ser Asn Ala Phe Leu Ile Asn Thr Leu Lys Lys Thr 130 135 140 Val Val Glu Ser Ala Ile Asn Asp Thr Leu Glu Ser Phe Arg Glu Asn 145 150 155 160 Leu Asn Asp Pro Arg Phe Asp Tyr Asn Lys Phe Phe Arg Lys Arg Met 165 170 175 Glu Ile Ile Tyr Asp Lys Gln Lys Asn Phe Ile Asn Tyr Tyr Lys Ala 180 185 190 Gln Arg Glu Glu Asn Pro Glu Leu Ile Ile Asp Asp Ile Val Lys Thr 195 200 205 Tyr Leu Ser Asn Glu Tyr Ser Lys Glu Ile Asp Glu Leu Asn Thr Tyr 210 215 220 Ile Glu Glu Ser Leu Asn Lys Ile Thr Gln Asn Ser Gly Asn Asp Val 225 230 235 240 Arg Asn Phe Glu Glu Phe Lys Asn Gly Glu Ser Phe Asn Leu Tyr Glu 245 250 255 Gln Glu Leu Val Glu Arg Trp Asn Leu Ala Ala Ala Ser Asp Ile Leu 260 265 270 Arg Ile Ser Ala Leu Lys Glu Ile Gly Gly Met Tyr Leu Asp Val Asp 275 280 285 Met Leu Pro Gly Ile Gln Pro Asp Leu Phe Glu Ser Ile Glu Lys Pro 290 295 300 Ser Ser Val Thr Val Asp Phe Trp Glu Met Thr Lys Leu Glu Ala Ile 305 310 315 320 Met Lys Tyr Lys Glu Tyr Ile Pro Glu Tyr Thr Ser Glu His Phe Asp 325 330 335 Met Leu Asp Glu Glu Val Gln Ser Ser Phe Glu Ser Val Leu Ala Ser 340 345 350 Lys Ser Asp Lys Ser Glu Ile Phe Ser Ser Leu Gly Asp Met Glu Ala 355 360 365 Ser Pro Leu Glu Val Lys Ile Ala Phe Asn Ser Lys Gly Ile Ile Asn 370 375 380 Gln Gly Leu Ile Ser Val Lys Asp Ser Tyr Cys Ser Asn Leu Ile Val 385 390 395 400 Lys Gln Ile Glu Asn Arg Tyr Lys Ile Leu Asn Asn Ser Leu Asn Pro 405 410 415 Ala Ile Ser Glu Asp Asn Asp Phe Asn Thr Thr Thr Asn Thr Phe Ile 420 425 430 Asp Ser Ile Met Ala Glu Ala Asn Ala Asp Asn Gly Arg Phe Met Met 435 440 445 Glu Leu Gly Lys Tyr Leu Arg Val Gly Phe Phe Pro Asp Val Lys Thr 450 455 460 Thr Ile Asn Leu Ser Gly Pro Glu Ala Tyr Ala Ala Ala Tyr Gln Asp 465 470 475 480 Leu Leu Met Phe Lys Glu Gly Ser Met Asn Ile His Leu Ile Glu Ala 485 490 495 Asp Leu Arg Asn Phe Glu Ile Ser Lys Thr Asn Ile Ser Gln Ser Thr 500 505 510 Glu Gln Glu Met Ala Ser Leu Trp Ser Phe Asp Asp Ala Arg Ala Lys 515 520 525 Ala Gln Phe Glu Glu Tyr Lys Arg Asn Tyr Phe Glu Gly Ser Leu Gly 530 535 540 Glu Asp Asp Asn Leu Asp Phe Ser Gln Asn Ile Val Val Asp Lys Glu 545 550 555 560 Tyr Leu Leu Glu Lys Ile Ser Ser Leu Ala Arg Ser Ser Glu Arg Gly 565 570 575 Tyr Ile His Tyr Ile Val Gln Leu Gln Gly Asp Lys Ile Ser Tyr Glu 580 585 590 Ala Ala Cys Asn Leu Phe Ala Lys Thr Pro Tyr Asp Ser Val Leu Phe 595 600 605 Gln Lys Asn Ile Glu Asp Ser Glu Ile Ala Tyr Tyr Tyr Asn Pro Gly 610 615 620 Asp Gly Glu Ile Gln Glu Ile Asp Lys Tyr Lys Ile Pro Ser Ile Ile 625 630 635 640 Ser Asp Arg Pro Lys Ile Lys Leu Thr Phe Ile Gly His Gly Lys Asp 645 650 655 Glu Phe Asn Thr Asp Ile Phe Ala Gly Phe Asp Val Asp Ser Leu Ser 660 665 670 Thr Glu Ile Glu Ala Ala Ile Asp Leu Ala Lys Glu Asp Ile Ser Pro 675 680 685 Lys Ser Ile Glu Ile Asn Leu Leu Gly Cys Asn Met Phe Ser Tyr Ser 690 695 700 Ile Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu Leu Leu Lys Val Lys 705 710 715 720 Asp Lys Ile Ser Glu Leu Met Pro Ser Ile Ser Gln Asp Ser Ile Ile 725 730 735 Val Ser Ala Asn Gln Tyr Glu Val Arg Ile Asn Ser Glu Gly Arg Arg 740 745 750 Glu Leu Leu Asp His Ser Gly Glu Trp Ile Asn Lys Glu Glu Ser Ile 755 760 765 Ile Lys Asp Ile Ser Ser Lys Glu Tyr Ile Ser Phe Asn Pro Lys Glu 770 775 780 Asn Lys Ile Thr Val Lys Ser Lys Asn Leu Pro Glu Leu Ser Thr Leu 785 790 795 800 Leu Gln Glu Ile Arg Asn Asn Ser Asn Ser Ser Asp Ile Glu Leu Glu 805 810 815 Glu Lys Val Met Leu Thr Glu Cys Glu Ile Asn Val Ile Ser Asn Ile 820 825 830 Asp Thr Gln Ile Val Glu Glu Arg Ile Glu Glu Ala Lys Asn Leu Thr 835 840 845 Ser Asp Ser Ile Asn Tyr Ile Lys Asp Glu Phe Lys Leu Ile Glu Ser 850 855 860 Ile Ser Asp Ala Leu Cys Asp Leu Lys Gln Gln Asn Glu Leu Glu Asp 865 870 875 880 Ser His Phe Ile Ser Phe Glu Asp Ile Ser Glu Thr Asp Glu Gly Phe 885 890 895 Ser Ile Arg Phe Ile Asn Lys Glu Thr Gly Glu Ser Ile Phe Val Glu 900 905 910 Thr Glu Lys Thr Ile Phe Ser Glu Tyr Ala Asn His Ile Thr Glu Glu 915 920 925 Ile Ser Lys Ile Lys Gly Thr Ile Phe Asp Thr Val Asn Gly Lys Leu 930 935 940 Val Lys Lys Val Asn Leu Asp Thr Thr His Glu Val Asn Thr Leu Asn 945 950 955 960 Ala Ala Phe Phe Ile Gln Ser Leu Ile Glu Tyr Asn Ser Ser Lys Glu 965 970 975 Ser Leu Ser Asn Leu Ser Val Ala Met Lys Val Gln Val Tyr Ala Gln 980 985 990 Leu Phe Ser Thr Gly Leu Asn Thr Ile Thr Asp Ala Ala Lys Val Val 995 1000 1005 Glu Leu Val Ser Thr Ala Leu Asp Glu Thr Ile Asp Leu Leu Pro Thr 1010 1015 1020 Leu Ser Glu Gly Leu Pro Ile Ile Ala Thr Ile Ile Asp Gly Val Ser 1025 1030 1035 1040 Leu Gly Ala Ala Ile Lys Glu Leu Ser Glu Thr Ser Asp Pro Leu Leu 1045 1050 1055 Arg Gln Glu Ile Glu Ala Lys Ile Gly Ile Met Ala Val Asn Leu Thr 1060 1065 1070 Thr Ala Thr Thr Ala Ile Ile Thr Ser Ser Leu Gly Ile Ala Ser Gly 1075 1080 1085 Phe Ser Ile Leu Leu Val Pro Leu Ala Gly Ile Ser Ala Gly Ile Pro 1090 1095 1100 Ser Leu Val Asn Asn Glu Leu Val Leu Arg Asp Lys Ala Thr Lys Val 1105 1110 1115 1120 Val Asp Tyr Phe Lys His Val Ser Leu Val Glu Thr Glu Gly Val Phe 1125 1130 1135 Thr Leu Leu Asp Asp Lys Ile Met Met Pro Gln Asp Asp Leu Val Ile 1140 1145 1150 Ser Glu Ile Asp Phe Asn Asn Asn Ser Ile Val Leu Gly Lys Cys Glu 1155 1160 1165 Ile Trp Arg Met Glu Gly Gly Ser Gly His Thr Val Thr Asp Asp Ile 1170 1175 1180 Asp His Phe Phe Ser Ala Pro Ser Ile Thr Tyr Arg Glu Pro His Leu 1185 1190 1195 1200 Ser Ile Tyr Asp Val Leu Glu Val Gln Lys Glu Glu Leu Asp Leu Ser 1205 1210 1215 Lys Asp Leu Met Val Leu Pro Asn Ala Pro Asn Arg Val Phe Ala Trp 1220 1225 1230 Glu Thr Gly Trp Thr Pro Gly Leu Arg Ser Leu Glu Asn Asp Gly Thr 1235 1240 1245 Lys Leu Leu Asp Arg Ile Arg Asp Asn Tyr Glu Gly Glu Phe Tyr Trp 1250 1255 1260 Arg Tyr Phe Ala Phe Ile Ala Asp Ala Leu Ile Thr Thr Leu Lys Pro 1265 1270 1275 1280 Arg Tyr Glu Asp Thr Asn Ile Arg Ile Asn Leu Asp Ser Asn Thr Arg 1285 1290 1295 Ser Phe Ile Val Pro Ile Ile Thr Thr Glu Tyr Ile Arg Glu Lys Leu 1300 1305 1310 Ser Tyr Ser Phe Tyr Gly Ser Gly Gly Thr Tyr Ala Leu Ser Leu Ser 1315 1320 1325 Gln Tyr Asn Met Gly Ile Asn Ile Glu Leu Ser Glu Ser Asp Val Trp 1330 1335 1340 Ile Ile Asp Val Asp Asn Val Val Arg Asp Val Thr Ile Glu Ser Asp 1345 1350 1355 1360 Lys Ile Lys Lys Gly Asp Leu Ile Glu Gly Ile Leu Ser Thr Leu Ser 1365 1370 1375 Ile Glu Glu Asn Lys Ile Ile Leu Asn Ser His Glu Ile Asn Phe Ser 1380 1385 1390 Gly Glu Val Asn Gly Ser Asn Gly Phe Val Ser Leu Thr Phe Ser Ile 1395 1400 1405 Leu Glu Gly Ile Asn Ala Ile Ile Glu Val Asp Leu Leu Ser Lys Ser 1410 1415 1420 Tyr Lys Leu Leu Ile Ser Gly Glu Leu Lys Ile Leu Met Leu Asn Ser 1425 1430 1435 1440 Asn His Ile Gln Gln Lys Ile Asp Tyr Ile Gly Phe Asn Ser Glu Leu 1445 1450 1455 Gln Lys Asn Ile Pro Tyr Ser Phe Val Asp Ser Glu Gly Lys Glu Asn 1460 1465 1470 Gly Phe Ile Asn Gly Ser Thr Lys Glu Gly Leu Phe Val Ser Glu Leu 1475 1480 1485 Pro Asp Val Val Leu Ile Ser Lys Val Tyr Met Asp Asp Ser Lys Pro 1490 1495 1500 Ser Phe Gly Tyr Tyr Ser Asn Asn Leu Lys Asp Val Lys Val Ile Thr 1505 1510 1515 1520 Lys Asp Asn Val Asn Ile Leu Thr Gly Tyr Tyr Leu Lys Asp Asp Ile 1525 1530 1535 Lys Ile Ser Leu Ser Leu Thr Leu Gln Asp Glu Lys Thr Ile Lys Leu 1540 1545 1550 Asn Ser Val His Leu Asp Glu Ser Gly Val Ala Glu Ile Leu Lys Phe 1555 1560 1565 Met Asn Arg Lys Gly Asn Thr Asn Thr Ser Asp Ser Leu Met Ser Phe 1570 1575 1580 Leu Glu Ser Met Asn Ile Lys Ser Ile Phe Val Asn Phe Leu Gln Ser 1585 1590 1595 1600 Asn Ile Lys Phe Ile Leu Asp Ala Asn Phe Ile Ile Ser Gly Thr Thr 1605 1610 1615 Ser Ile Gly Gln Phe Glu Phe Ile Cys Asp Glu Asn Asp Asn Ile Gln 1620 1625 1630 Pro Tyr Phe Ile Lys Phe Asn Thr Leu Glu Thr Asn Tyr Thr Leu Tyr 1635 1640 1645 Val Gly Asn Arg Gln Asn Met Ile Val Glu Pro Asn Tyr Asp Leu Asp 1650 1655 1660 Asp Ser Gly Asp Ile Ser Ser Thr Val Ile Asn Phe Ser Gln Lys Tyr 1665 1670 1675 1680 Leu Tyr Gly Ile Asp Ser Cys Val Asn Lys Val Val Ile Ser Pro Asn 1685 1690 1695 Ile Tyr Thr Asp Glu Ile Asn Ile Thr Pro Val Tyr Glu Thr Asn Asn 1700 1705 1710 Thr Tyr Pro Glu Val Ile Val Leu Asp Ala Asn Tyr Ile Asn Glu Lys 1715 1720 1725 Ile Asn Val Asn Ile Asn Asp Leu Ser Ile Arg Tyr Val Trp Ser Asn 1730 1735 1740 Asp Gly Asn Asp Phe Ile Leu Met Ser Thr Ser Glu Glu Asn Lys Val 1745 1750 1755 1760 Ser Gln Val Lys Ile Arg Phe Val Asn Val Phe Lys Asp Lys Thr Leu 1765 1770 1775 Ala Asn Lys Leu Ser Phe Asn Phe Ser Asp Lys Gln Asp Val Pro Val 1780 1785 1790 Ser Glu Ile Ile Leu Ser Phe Thr Pro Ser Tyr Tyr Glu Asp Gly Leu 1795 1800 1805 Ile Gly Tyr Asp Leu Gly Leu Val Ser Leu Tyr Asn Glu Lys Phe Tyr 1810 1815 1820 Ile Asn Asn Phe Gly Met Met Val Ser Gly Leu Ile Tyr Ile Asn Asp 1825 1830 1835 1840 Ser Leu Tyr Tyr Phe Lys Pro Pro Val Asn Asn Leu Ile Thr Gly Phe 1845 1850 1855 Val Thr Val Gly Asp Asp Lys Tyr Tyr Phe Asn Pro Ile Asn Gly Gly 1860 1865 1870 Ala Ala Ser Ile Gly Glu Thr Ile Ile Asp Asp Lys Asn Tyr Tyr Phe 1875 1880 1885 Asn Gln Ser Gly Val Leu Gln Thr Gly Val Phe Ser Thr Glu Asp Gly 1890 1895 1900 Phe Lys Tyr Phe Ala Pro Ala Asn Thr Leu Asp Glu Asn Leu Glu Gly 1905 1910 1915 1920 Glu Ala Ile Asp Phe Thr Gly Lys Leu Ile Ile Asp Glu Asn Ile Tyr 1925 1930 1935 Tyr Phe Asp Asp Asn Tyr Arg Gly Ala Val Glu Trp Lys Glu Leu Asp 1940 1945 1950 Gly Glu Met His Tyr Phe Ser Pro Glu Thr Gly Lys Ala Phe Lys Gly 1955 1960 1965 Leu Asn Gln Ile Gly Asp Tyr Lys Tyr Tyr Phe Asn Ser Asp Gly Val 1970 1975 1980 Met Gln Lys Gly Phe Val Ser Ile Asn Asp Asn Lys His Tyr Phe Asp 1985 1990 1995 2000 Asp Ser Gly Val Met Lys Val Gly Tyr Thr Glu Ile Asp Gly Lys His 2005 2010 2015 Phe Tyr Phe Ala Glu Asn Gly Glu Met Gln Ile Gly Val Phe Asn Thr 2020 2025 2030 Glu Asp Gly Phe Lys Tyr Phe Ala His His Asn Glu Asp Leu Gly Asn 2035 2040 2045 Glu Glu Gly Glu Glu Ile Ser Tyr Ser Gly Ile Leu Asn Phe Asn Asn 2050 2055 2060 Lys Ile Tyr Tyr Phe Asp Asp Ser Phe Thr Ala Val Val Gly Trp Lys 2065 2070 2075 2080 Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe Asp Glu Asp Thr Ala Glu 2085 2090 2095 Ala Tyr Ile Gly Leu Ser Leu Ile Asn Asp Gly Gln Tyr Tyr Phe Asn 2100 2105 2110 Asp Asp Gly Ile Met Gln Val Gly Phe Val Thr Ile Asn Asp Lys Val 2115 2120 2125 Phe Tyr Phe Ser Asp Ser Gly Ile Ile Glu Ser Gly Val Gln Asn Ile 2130 2135 2140 Asp Asp Asn Tyr Phe Tyr Ile Asp Asp Asn Gly Ile Val Gln Ile Gly 2145 2150 2155 2160 Val Phe Asp Thr Ser Asp Gly Tyr Lys Tyr Phe Ala Pro Ala Asn Thr 2165 2170 2175 Val Asn Asp Asn Ile Tyr Gly Gln Ala Val Glu Tyr Ser Gly Leu Val 2180 2185 2190 Arg Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Thr Ile Glu 2195 2200 2205 Thr Gly Trp Ile Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr Tyr Phe 2210 2215 2220 Asn Pro Glu Thr Lys Lys Ala Cys Lys Gly Ile Asn Leu Ile Asp Asp 2225 2230 2235 2240 Ile Lys Tyr Tyr Phe Asp Glu Lys Gly Ile Met Arg Thr Gly Leu Ile 2245 2250 2255 Ser Phe Glu Asn Asn Asn Tyr Tyr Phe Asn Glu Asn Gly Glu Met Gln 2260 2265 2270 Phe Gly Tyr Ile Asn Ile Glu Asp Lys Met Phe Tyr Phe Gly Glu Asp 2275 2280 2285 Gly Val Met Gln Ile Gly Val Phe Asn Thr Pro Asp Gly Phe Lys Tyr 2290 2295 2300 Phe Ala His Gln Asn Thr Leu Asp Glu Asn Phe Glu Gly Glu Ser Ile 2305 2310 2315 2320 Asn Tyr Thr Gly Trp Leu Asp Leu Asp Glu Lys Arg Tyr Tyr Phe Thr 2325 2330 2335 Asp Glu Tyr Ile Ala Ala Thr Gly Ser Val Ile Ile Asp Gly Glu Glu 2340 2345 2350 Tyr Tyr Phe Asp Pro Asp Thr Ala Gln Leu Val Ile Ser Glu 2355 2360 2365

11 TAGAAAAAAT GGCAAATGT 19

12 TTTCATCTTG TAGAGTCAAA G 21

13 GATGCCACAA GATGATTTAG TG 22

14 CTAATTGAGC TGTATCAGGA TC 22

15 CGGAATTCCT AGAAAAAATG GCAAATG 27

16 GCTCTAGAAT GACCATAAGC TAGCCA 26

17 CGGAATTCGA GTTGGTAGAA AGGTGGA 27

18 CGGAATTCGG TTATTATCTT AAGGATG 27

19 CGGAATTCTT GATAACTGGA TTTGTGAC 28

20 Leu Ile Thr Gly Phe Val Thr Val Gly Asp Asp Lys Tyr Tyr Phe Asn 1 5 10 15 Pro Ile Asn Gly Gly Ala Ala Ser Ile Gly Glu Thr Ile Ile Asp Asp 20 25 30 Lys Asn Tyr Tyr Phe Asn Gln Ser Gly Val Leu Gln Thr Gly Val Phe 35 40 45 Ser Thr Glu Asp Gly Phe Lys Tyr Phe Ala Pro Ala Asn Thr Leu Asp 50 55 60 Glu Asn Leu Glu Gly Glu Ala Ile Asp Phe Thr Gly Lys Leu Ile Ile 65 70 75 80 Asp Glu Asn Ile Tyr Tyr Phe Asp Asp Asn Tyr Arg Gly Ala Val Glu 85 90 95 Trp Lys Glu Leu Asp Gly Glu Met His Tyr Phe Ser Pro Glu Thr Gly 100 105 110 Lys Ala Phe Lys Gly Leu Asn Gln Ile Gly Asp Tyr Lys Tyr Tyr Phe 115 120 125 Asn Ser Asp Gly Val Met Gln Lys Gly Phe Val Ser Ile Asn Asp Asn 130 135 140 Lys His Tyr Phe Asp Asp Ser Gly Val Met Lys Val Gly Tyr Thr Glu 145 150 155 160 Ile Asp Gly Lys His Phe Tyr Phe Ala Glu Asn Gly Glu Met Gln Ile 165 170 175 Gly Val Phe Asn Thr Glu Asp Gly Phe Lys Tyr Phe Ala His His Asn 180 185 190 Glu Asp Leu Gly Asn Glu Glu Gly Glu Glu Ile Ser Tyr Ser Gly Ile 195 200 205 Leu Asn Phe Asn Asn Lys Ile Tyr Tyr Phe Asp Asp Ser Phe Thr Ala 210 215 220 Val Val Gly Trp Lys Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe Asp 225 230 235 240 Glu Asp Thr Ala Glu Ala Tyr Ile Gly Leu Ser Leu Ile Asn Asp Gly 245 250 255 Gln Tyr Tyr Phe Asn Asp Asp Gly Ile Met Gln Val Gly Phe Val Thr 260 265 270 Ile Asn Asp Lys Val Phe Tyr Phe Ser Asp Ser Gly Ile Ile Glu Ser 275 280 285 Gly Val Gln Asn Ile Asp Asp Asn Tyr Phe Tyr Ile Asp Asp Asn Gly 290 295 300 Ile Val Gln Ile Gly Val Phe Asp Thr Ser Asp Gly Tyr Lys Tyr Phe 305 310 315 320 Ala Pro Ala Asn Thr Val Asn Asp Asn Ile Tyr Gly Gln Ala Val Glu 325 330 335 Tyr Ser Gly Leu Val Arg Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu 340 345 350 Thr Tyr Thr Ile Glu Thr Gly Trp Ile Tyr Asp Met Glu Asn Glu Ser 355 360 365 Asp Lys Tyr Tyr Phe Asn Pro Glu Thr Lys Lys Ala Cys Lys Gly Ile 370 375 380 Asn Leu Ile Asp Asp Ile Lys Tyr Tyr Phe Asp Glu Lys Gly Ile Met 385 390 395 400 Arg Thr Gly Leu Ile Ser Phe Glu Asn Asn Asn Tyr Tyr Phe Asn Glu 405 410 415 Asn Gly Glu Met Gln Phe Gly Tyr Ile Asn Ile Glu Asp Lys Met Phe 420 425 430 Tyr Phe Gly Glu Asp Gly Val Met Gln Ile Gly Val Phe Asn Thr Pro 435 440 445 Asp Gly Phe Lys Tyr Phe Ala His Gln Asn Thr Leu Asp Glu Asn Phe 450 455 460 Glu Gly Glu Ser Ile Asn Tyr Thr Gly Trp Leu Asp Leu Asp Glu Lys 465 470 475 480 Arg Tyr Tyr Phe Thr Asp Glu Tyr Ile Ala Ala Thr Gly Ser Val Ile 485 490 495 Ile Asp Gly Glu Glu Tyr Tyr Phe Asp Pro Asp Thr Ala Gln Leu 500 505 510

21 Ser Glu Glu Asn Lys Val Ser Gln Val Lys Ile Arg Phe Val Asn Val 1 5 10 15 Phe Lys Asp Lys Thr Leu Ala Asn Lys Leu Ser Phe Asn Phe Ser Asp 20 25 30 Lys Gln Asp Val Pro Val Ser Glu Ile Ile Leu Ser Phe Thr Pro Ser 35 40 45 Tyr Tyr Glu Asp Gly Leu Ile Gly Tyr Asp Leu Gly Leu Val Ser Leu 50 55 60 Tyr Asn Glu Lys Phe Tyr Ile Asn Asn Phe Gly Met Met Val Ser Gly 65 70 75 80 Leu Ile Tyr Ile Asn Asp Ser Leu Tyr Tyr Phe Lys Pro Pro Val Asn 85 90 95 Asn Leu Ile Thr Gly Phe Val Thr Val Gly Asp Asp Lys Tyr Tyr Phe 100 105 110 Asn Pro Ile Asn Gly Gly Ala Ala Ser Ile Gly Glu Thr Ile Ile Asp 115 120 125 Asp Lys Asn Tyr Tyr Phe Asn Gln Ser Gly Val Leu Gln Thr Gly Val 130 135 140 Phe Ser Thr Glu Asp Gly Phe Lys Tyr Phe Ala Pro Ala Asn Thr Leu 145 150 155 160 Asp Glu Asn Leu Glu Gly Glu Ala Ile Asp Phe Thr Gly Lys Leu Ile 165 170 175 Ile Asp Glu Asn Ile Tyr Tyr Phe Asp Asp Asn Tyr Arg Gly Ala Val 180 185 190 Glu Trp Lys Glu Leu Asp Gly Glu Met His Tyr Phe Ser Pro Glu Thr 195 200 205 Gly Lys Ala Phe Lys Gly Leu Asn Gln Ile Gly Asp Tyr Lys Tyr Tyr 210 215 220 Phe Asn Ser Asp Gly Val Met Gln Lys Gly Phe Val Ser Ile Asn Asp 225 230 235 240 Asn Lys His Tyr Phe Asp Asp Ser Gly Val Met Lys Val Gly Tyr Thr 245 250 255 Glu Ile Asp Gly Lys His Phe Tyr Phe Ala Glu Asn Gly Glu Met Gln 260 265 270 Ile Gly Val Phe Asn Thr Glu Asp Gly Phe Lys Tyr Phe Ala His His 275 280 285 Asn Glu Asp Leu Gly Asn Glu Glu Gly Glu Glu Ile Ser Tyr Ser Gly 290 295 300 Ile Leu Asn Phe Asn Asn Lys Ile Tyr Tyr Phe Asp Asp Ser Phe Thr 305 310 315 320 Ala Val Val Gly Trp Lys Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe 325 330 335 Asp Glu Asp Thr Ala Glu Ala Tyr Ile Gly Leu Ser Leu Ile Asn Asp 340 345 350 Gly Gln Tyr Tyr Phe Asn Asp Asp Gly Ile Met Gln Val Gly Phe Val 355 360 365 Thr Ile Asn Asp Lys Val Phe Tyr Phe Ser Asp Ser Gly Ile Ile Glu 370 375 380 Ser Gly Val Gln Asn Ile Asp Asp Asn Tyr Phe Tyr Ile Asp Asp Asn 385 390 395 400 Gly Ile Val Gln Ile Gly Val Phe Asp Thr Ser Asp Gly Tyr Lys Tyr 405 410 415 Phe Ala Pro Ala Asn Thr Val Asn Asp Asn Ile Tyr Gly Gln Ala Val 420 425 430 Glu Tyr Ser Gly Leu Val Arg Val Gly Glu Asp Val Tyr Tyr Phe Gly 435 440 445 Glu Thr Tyr Thr Ile Glu Thr Gly Trp Ile Tyr Asp Met Glu Asn Glu 450 455 460 Ser Asp Lys Tyr Tyr Phe Asn Pro Glu Thr Lys Lys Ala Cys Lys Gly 465 470 475 480 Ile Asn Leu Ile Asp Asp Ile Lys Tyr Tyr Phe Asp Glu Lys Gly Ile 485 490 495 Met Arg Thr Gly Leu Ile Ser Phe Glu Asn Asn Asn Tyr Tyr Phe Asn 500 505 510 Glu Asn Gly Glu Met Gln Phe Gly Tyr Ile Asn Ile Glu Asp Lys Met 515 520 525 Phe Tyr Phe Gly Glu Asp Gly Val Met Gln Ile Gly Val Phe Asn Thr 530 535 540 Pro Asp Gly Phe Lys Tyr Phe Ala His Gln Asn Thr Leu Asp Glu Asn 545 550 555 560 Phe Glu Gly Glu Ser Ile Asn Tyr Thr Gly Trp Leu Asp Leu Asp Glu 565 570 575 Lys Arg Tyr Tyr Phe Thr Asp Glu Tyr Ile Ala Ala Thr Gly Ser Val 580 585 590 Ile Ile Asp Gly Glu Glu Tyr Tyr Phe Asp Pro Asp Thr Ala Gln Leu 595 600 605

22 ATG GCT CGT CTG CTG TCT ACC TTC ACT GAA TAC ATC AAG AAC ATC ATC 48 Met Ala Arg Leu Leu Ser Thr Phe Thr Glu Tyr Ile Lys Asn Ile Ile 1 5 10 15 AAT ACC TCC ATC CTG AAC CTG CGC TAC GAA TCC AAT CAC CTG ATC GAC 96 Asn Thr Ser Ile Leu Asn Leu Arg Tyr Glu Ser Asn His Leu Ile Asp 20 25 30 CTG TCT CGC TAC GCT TCC AAA ATC AAC ATC GGT TCT AAA GTT AAC TTC 144 Leu Ser Arg Tyr Ala Ser Lys Ile Asn Ile Gly Ser Lys Val Asn Phe 35 40 45 GAT CCG ATC GAC AAG AAT CAG ATC CAG CTG TTC AAT CTG GAA TCT TCC 192 Asp Pro Ile Asp Lys Asn Gln Ile Gln Leu Phe Asn Leu Glu Ser Ser 50 55 60 AAA ATC GAA GTT ATC CTG AAG AAT GCT ATC GTA TAC AAC TCT ATG TAC 240 Lys Ile Glu Val Ile Leu Lys Asn Ala Ile Val Tyr Asn Ser Met Tyr 65 70 75 80 GAA AAC TTC TCC ACC TCC TTC TGG ATC CGT ATC CCG AAA TAC TTC AAC 288 Glu Asn Phe Ser Thr Ser Phe Trp Ile Arg Ile Pro Lys Tyr Phe Asn 85 90 95 TCC ATC TCT CTG AAC AAT GAA TAC ACC ATC ATC AAC TGC ATG GAA AAC 336 Ser Ile Ser Leu Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Glu Asn 100 105 110 AAT TCT GGT TGG AAA GTA TCT CTG AAC TAC GGT GAA ATC ATC TGG ACT 384 Asn Ser Gly Trp Lys Val Ser Leu Asn Tyr Gly Glu Ile Ile Trp Thr 115 120 125 CTG CAG GAC ACT CAG GAA ATC AAA CAG CGT GTT GTA TTC AAA TAC TCT 432 Leu Gln Asp Thr Gln Glu Ile Lys Gln Arg Val Val Phe Lys Tyr Ser 130 135 140 CAG ATG ATC AAC ATC TCT GAC TAC ATC AAT CGC TGG ATC TTC GTT ACC 480 Gln Met Ile Asn Ile Ser Asp Tyr Ile Asn Arg Trp Ile Phe Val Thr 145 150 155 160 ATC ACC AAC AAT CGT CTG AAT AAC TCC AAA ATC TAC ATC AAC GGC CGT 528 Ile Thr Asn Asn Arg Leu Asn Asn Ser Lys Ile Tyr Ile Asn Gly Arg 165 170 175 CTG ATC GAC CAG AAA CCG ATC TCC AAT CTG GGT AAC ATC CAC GCT TCT 576 Leu Ile Asp Gln Lys Pro Ile Ser Asn Leu Gly Asn Ile His Ala Ser 180 185 190 AAT AAC ATC ATG TTC AAA CTG GAC GGT TGT CGT GAC ACT CAC CGC TAC 624 Asn Asn Ile Met Phe Lys Leu Asp Gly Cys Arg Asp Thr His Arg Tyr 195 200 205 ATC TGG ATC AAA TAC TTC AAT CTG TTC GAC AAA GAA CTG AAC GAA AAA 672 Ile Trp Ile Lys Tyr Phe Asn Leu Phe Asp Lys Glu Leu Asn Glu Lys 210 215 220 GAA ATC AAA GAC CTG TAC GAC AAC CAG TCC AAT TCT GGT ATC CTG AAA 720 Glu Ile Lys Asp Leu Tyr Asp Asn Gln Ser Asn Ser Gly Ile Leu Lys 225 230 235 240 GAC TTC TGG GGT GAC TAC CTG CAG TAC GAC AAA CCG TAC TAC ATG CTG 768 Asp Phe Trp Gly Asp Tyr Leu Gln Tyr Asp Lys Pro Tyr Tyr Met Leu 245 250 255 AAT CTG TAC GAT CCG AAC AAA TAC GTT GAC GTC AAC AAT GTA GGT ATC 816 Asn Leu Tyr Asp Pro Asn Lys Tyr Val Asp Val Asn Asn Val Gly Ile 260 265 270 CGC GGT TAC ATG TAC CTG AAA GGT CCG CGT GGT TCT GTT ATG ACT ACC 864 Arg Gly Tyr Met Tyr Leu Lys Gly Pro Arg Gly Ser Val Met Thr Thr 275 280 285 AAC ATC TAC CTG AAC TCT TCC CTG TAC CGT GGT ACC AAA TTC ATC ATC 912 Asn Ile Tyr Leu Asn Ser Ser Leu Tyr Arg Gly Thr Lys Phe Ile Ile 290 295 300 AAG AAA TAC GCG TCT GGT AAC AAG GAC AAT ATC GTT CGC AAC AAT GAT 960 Lys Lys Tyr Ala Ser Gly Asn Lys Asp Asn Ile Val Arg Asn Asn Asp 305 310 315 320 CGT GTA TAC ATC AAT GTT GTA GTT AAG AAC AAA GAA TAC CGT CTG GCT 1008 Arg Val Tyr Ile Asn Val Val Val Lys Asn Lys Glu Tyr Arg Leu Ala 325 330 335 ACC AAT GCT TCT CAG GCT GGT GTA GAA AAG ATC TTG TCT GCT CTG GAA 1056 Thr Asn Ala Ser Gln Ala Gly Val Glu Lys Ile Leu Ser Ala Leu Glu 340 345 350 ATC CCG GAC GTT GGT AAT CTG TCT CAG GTA GTT GTA ATG AAA TCC AAG 1104 Ile Pro Asp Val Gly Asn Leu Ser Gln Val Val Val Met Lys Ser Lys 355 360 365 AAC GAC CAG GGT ATC ACT AAC AAA TGC AAA ATG AAT CTG CAG GAC AAC 1152 Asn Asp Gln Gly Ile Thr Asn Lys Cys Lys Met Asn Leu Gln Asp Asn 370 375 380 AAT GGT AAC GAT ATC GGT TTC ATC GGT TTC CAC CAG TTC AAC AAT ATC 1200 Asn Gly Asn Asp Ile Gly Phe Ile Gly Phe His Gln Phe Asn Asn Ile 385 390 395 400 GCT AAA CTG GTT GCT TCC AAC TGG TAC AAT CGT CAG ATC GAA CGT TCC 1248 Ala Lys Leu Val Ala Ser Asn Trp Tyr Asn Arg Gln Ile Glu Arg Ser 405 410 415 TCT CGC ACT CTG GGT TGC TCT TGG GAG TTC ATC CCG GTT GAT GAC GGT 1296 Ser Arg Thr Leu Gly Cys Ser Trp Glu Phe Ile Pro Val Asp Asp Gly 420 425 430 TGG GGT GAA CGT CCG CTG TAACCCGGGA AAGCTT 1330 Trp Gly Glu Arg Pro Leu 435

23 Met Ala Arg Leu Leu Ser Thr Phe Thr Glu Tyr Ile Lys Asn Ile Ile 1 5 10 15 Asn Thr Ser Ile Leu Asn Leu Arg Tyr Glu Ser Asn His Leu Ile Asp 20 25 30 Leu Ser Arg Tyr Ala Ser Lys Ile Asn Ile Gly Ser Lys Val Asn Phe 35 40 45 Asp Pro Ile Asp Lys Asn Gln Ile Gln Leu Phe Asn Leu Glu Ser Ser 50 55 60 Lys Ile Glu Val Ile Leu Lys Asn Ala Ile Val Tyr Asn Ser Met Tyr 65 70 75 80 Glu Asn Phe Ser Thr Ser Phe Trp Ile Arg Ile Pro Lys Tyr Phe Asn 85 90 95 Ser Ile Ser Leu Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Glu Asn 100 105 110 Asn Ser Gly Trp Lys Val Ser Leu Asn Tyr Gly Glu Ile Ile Trp Thr 115 120 125 Leu Gln Asp Thr Gln Glu Ile Lys Gln Arg Val Val Phe Lys Tyr Ser 130 135 140 Gln Met Ile Asn Ile Ser Asp Tyr Ile Asn Arg Trp Ile Phe Val Thr 145 150 155 160 Ile Thr Asn Asn Arg Leu Asn Asn Ser Lys Ile Tyr Ile Asn Gly Arg 165 170 175 Leu Ile Asp Gln Lys Pro Ile Ser Asn Leu Gly Asn Ile His Ala Ser 180 185 190 Asn Asn Ile Met Phe Lys Leu Asp Gly Cys Arg Asp Thr His Arg Tyr 195 200 205 Ile Trp Ile Lys Tyr Phe Asn Leu Phe Asp Lys Glu Leu Asn Glu Lys 210 215 220 Glu Ile Lys Asp Leu Tyr Asp Asn Gln Ser Asn Ser Gly Ile Leu Lys 225 230 235 240 Asp Phe Trp Gly Asp Tyr Leu Gln Tyr Asp Lys Pro Tyr Tyr Met Leu 245 250 255 Asn Leu Tyr Asp Pro Asn Lys Tyr Val Asp Val Asn Asn Val Gly Ile 260 265 270 Arg Gly Tyr Met Tyr Leu Lys Gly Pro Arg Gly Ser Val Met Thr Thr 275 280 285 Asn Ile Tyr Leu Asn Ser Ser Leu Tyr Arg Gly Thr Lys Phe Ile Ile 290 295 300 Lys Lys Tyr Ala Ser Gly Asn Lys Asp Asn Ile Val Arg Asn Asn Asp 305 310 315 320 Arg Val Tyr Ile Asn Val Val Val Lys Asn Lys Glu Tyr Arg Leu Ala 325 330 335 Thr Asn Ala Ser Gln Ala Gly Val Glu Lys Ile Leu Ser Ala Leu Glu 340 345 350 Ile Pro Asp Val Gly Asn Leu Ser Gln Val Val Val Met Lys Ser Lys 355 360 365 Asn Asp Gln Gly Ile Thr Asn Lys Cys Lys Met Asn Leu Gln Asp Asn 370 375 380 Asn Gly Asn Asp Ile Gly Phe Ile Gly Phe His Gln Phe Asn Asn Ile 385 390 395 400 Ala Lys Leu Val Ala Ser Asn Trp Tyr Asn Arg Gln Ile Glu Arg Ser 405 410 415 Ser Arg Thr Leu Gly Cys Ser Trp Glu Phe Ile Pro Val Asp Asp Gly 420 425 430 Trp Gly Glu Arg Pro Leu 435

24 Met Gly His His His His His His His His His His Ser Ser Gly His 1 5 10 15 Ile Glu Gly Arg His Met Ala 20

25 ATG GGC CAT CAT CAT CAT CAT CAT CAT CAT CAT CAC AGC AGC GGC CAT 48 Met Gly His His His His His His His His His His Ser Ser Gly His 1 5 10 15 ATC GAA GGT CGT CAT ATG GCT AGC ATG GCT CGT CTG CTG TCT ACC TTC 96 Ile Glu Gly Arg His Met Ala Ser Met Ala Arg Leu Leu Ser Thr Phe 20 25 30 ACT GAA TAC ATC AAG AAC ATC ATC AAT ACC TCC ATC CTG AAC CTG CGC 144 Thr Glu Tyr Ile Lys Asn Ile Ile Asn Thr Ser Ile Leu Asn Leu Arg 35 40 45 TAC GAA TCC AAT CAC CTG ATC GAC CTG TCT CGC TAC GCT TCC AAA ATC 192 Tyr Glu Ser Asn His Leu Ile Asp Leu Ser Arg Tyr Ala Ser Lys Ile 50 55 60 AAC ATC GGT TCT AAA GTT AAC TTC GAT CCG ATC GAC AAG AAT CAG ATC 240 Asn Ile Gly Ser Lys Val Asn Phe Asp Pro Ile Asp Lys Asn Gln Ile 65 70 75 80 CAG CTG TTC AAT CTG GAA TCT TCC AAA ATC GAA GTT ATC CTG AAG AAT 288 Gln Leu Phe Asn Leu Glu Ser Ser Lys Ile Glu Val Ile Leu Lys Asn 85 90 95 GCT ATC GTA TAC AAC TCT ATG TAC GAA AAC TTC TCC ACC TCC TTC TGG 336 Ala Ile Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser Phe Trp 100 105 110 ATC CGT ATC CCG AAA TAC TTC AAC TCC ATC TCT CTG AAC AAT GAA TAC 384 Ile Arg Ile Pro Lys Tyr Phe Asn Ser Ile Ser Leu Asn Asn Glu Tyr 115 120 125 ACC ATC ATC AAC TGC ATG GAA AAC AAT TCT GGT TGG AAA GTA TCT CTG 432 Thr Ile Ile Asn Cys Met Glu Asn Asn Ser Gly Trp Lys Val Ser Leu 130 135 140 AAC TAC GGT GAA ATC ATC TGG ACT CTG CAG GAC ACT CAG GAA ATC AAA 480 Asn Tyr Gly Glu Ile Ile Trp Thr Leu Gln Asp Thr Gln Glu Ile Lys 145 150 155 160 CAG CGT GTT GTA TTC AAA TAC TCT CAG ATG ATC AAC ATC TCT GAC TAC 528 Gln Arg Val Val Phe Lys Tyr Ser Gln Met Ile Asn Ile Ser Asp Tyr 165 170 175 ATC AAT CGC TGG ATC TTC GTT ACC ATC ACC AAC AAT CGT CTG AAT AAC 576 Ile Asn Arg Trp Ile Phe Val Thr Ile Thr Asn Asn Arg Leu Asn Asn 180 185 190 TCC AAA ATC TAC ATC AAC GGC CGT CTG ATC GAC CAG AAA CCG ATC TCC 624 Ser Lys Ile Tyr Ile Asn Gly Arg Leu Ile Asp Gln Lys Pro Ile Ser 195 200 205 AAT CTG GGT AAC ATC CAC GCT TCT AAT AAC ATC ATG TTC AAA CTG GAC 672 Asn Leu Gly Asn Ile His Ala Ser Asn Asn Ile Met Phe Lys Leu Asp 210 215 220 GGT TGT CGT GAC ACT CAC CGC TAC ATC TGG ATC AAA TAC TTC AAT CTG 720 Gly Cys Arg Asp Thr His Arg Tyr Ile Trp Ile Lys Tyr Phe Asn Leu 225 230 235 240 TTC GAC AAA GAA CTG AAC GAA AAA GAA ATC AAA GAC CTG TAC GAC AAC 768 Phe Asp Lys Glu Leu Asn Glu Lys Glu Ile Lys Asp Leu Tyr Asp Asn 245 250 255 CAG TCC AAT TCT GGT ATC CTG AAA GAC TTC TGG GGT GAC TAC CTG CAG 816 Gln Ser Asn Ser Gly Ile Leu Lys Asp Phe Trp Gly Asp Tyr Leu Gln 260 265 270 TAC GAC AAA CCG TAC TAC ATG CTG AAT CTG TAC GAT CCG AAC AAA TAC 864 Tyr Asp Lys Pro Tyr Tyr Met Leu Asn Leu Tyr Asp Pro Asn Lys Tyr 275 280 285 GTT GAC GTC AAC AAT GTA GGT ATC CGC GGT TAC ATG TAC CTG AAA GGT 912 Val Asp Val Asn Asn Val Gly Ile Arg Gly Tyr Met Tyr Leu Lys Gly 290 295 300 CCG CGT GGT TCT GTT ATG ACT ACC AAC ATC TAC CTG AAC TCT TCC CTG 960 Pro Arg Gly Ser Val Met Thr Thr Asn Ile Tyr Leu Asn Ser Ser Leu 305 310 315 320 TAC CGT GGT ACC AAA TTC ATC ATC AAG AAA TAC GCG TCT GGT AAC AAG 1008 Tyr Arg Gly Thr Lys Phe Ile Ile Lys Lys Tyr Ala Ser Gly Asn Lys 325 330 335 GAC AAT ATC GTT CGC AAC AAT GAT CGT GTA TAC ATC AAT GTT GTA GTT 1056 Asp Asn Ile Val Arg Asn Asn Asp Arg Val Tyr Ile Asn Val Val Val 340 345 350 AAG AAC AAA GAA TAC CGT CTG GCT ACC AAT GCT TCT CAG GCT GGT GTA 1104 Lys Asn Lys Glu Tyr Arg Leu Ala Thr Asn Ala Ser Gln Ala Gly Val 355 360 365 GAA AAG ATC TTG TCT GCT CTG GAA ATC CCG GAC GTT GGT AAT CTG TCT 1152 Glu Lys Ile Leu Ser Ala Leu Glu Ile Pro Asp Val Gly Asn Leu Ser 370 375 380 CAG GTA GTT GTA ATG AAA TCC AAG AAC GAC CAG GGT ATC ACT AAC AAA 1200 Gln Val Val Val Met Lys Ser Lys Asn Asp Gln Gly Ile Thr Asn Lys 385 390 395 400 TGC AAA ATG AAT CTG CAG GAC AAC AAT GGT AAC GAT ATC GGT TTC ATC 1248 Cys Lys Met Asn Leu Gln Asp Asn Asn Gly Asn Asp Ile Gly Phe Ile 405 410 415 GGT TTC CAC CAG TTC AAC AAT ATC GCT AAA CTG GTT GCT TCC AAC TGG 1296 Gly Phe His Gln Phe Asn Asn Ile Ala Lys Leu Val Ala Ser Asn Trp 420 425 430 TAC AAT CGT CAG ATC GAA CGT TCC TCT CGC ACT CTG GGT TGC TCT TGG 1344 Tyr Asn Arg Gln Ile Glu Arg Ser Ser Arg Thr Leu Gly Cys Ser Trp 435 440 445 GAG TTC ATC CCG GTT GAT GAC GGT TGG GGT GAA CGT CCG CTG 1386 Glu Phe Ile Pro Val Asp Asp Gly Trp Gly Glu Arg Pro Leu 450 455 460 TAACCCGGGA AAGCTT 1402

26 Met Gly His His His His His His His His His His Ser Ser Gly His 1 5 10 15 Ile Glu Gly Arg His Met Ala Ser Met Ala Arg Leu Leu Ser Thr Phe 20 25 30 Thr Glu Tyr Ile Lys Asn Ile Ile Asn Thr Ser Ile Leu Asn Leu Arg 35 40 45 Tyr Glu Ser Asn His Leu Ile Asp Leu Ser Arg Tyr Ala Ser Lys Ile 50 55 60 Asn Ile Gly Ser Lys Val Asn Phe Asp Pro Ile Asp Lys Asn Gln Ile 65 70 75 80 Gln Leu Phe Asn Leu Glu Ser Ser Lys Ile Glu Val Ile Leu Lys Asn 85 90 95 Ala Ile Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser Phe Trp 100 105 110 Ile Arg Ile Pro Lys Tyr Phe Asn Ser Ile Ser Leu Asn Asn Glu Tyr 115 120 125 Thr Ile Ile Asn Cys Met Glu Asn Asn Ser Gly Trp Lys Val Ser Leu 130 135 140 Asn Tyr Gly Glu Ile Ile Trp Thr Leu Gln Asp Thr Gln Glu Ile Lys 145 150 155 160 Gln Arg Val Val Phe Lys Tyr Ser Gln Met Ile Asn Ile Ser Asp Tyr 165 170 175 Ile Asn Arg Trp Ile Phe Val Thr Ile Thr Asn Asn Arg Leu Asn Asn 180 185 190 Ser Lys Ile Tyr Ile Asn Gly Arg Leu Ile Asp Gln Lys Pro Ile Ser 195 200 205 Asn Leu Gly Asn Ile His Ala Ser Asn Asn Ile Met Phe Lys Leu Asp 210 215 220 Gly Cys Arg Asp Thr His Arg Tyr Ile Trp Ile Lys Tyr Phe Asn Leu 225 230 235 240 Phe Asp Lys Glu Leu Asn Glu Lys Glu Ile Lys Asp Leu Tyr Asp Asn 245 250 255 Gln Ser Asn Ser Gly Ile Leu Lys Asp Phe Trp Gly Asp Tyr Leu Gln 260 265 270 Tyr Asp Lys Pro Tyr Tyr Met Leu Asn Leu Tyr Asp Pro Asn Lys Tyr 275 280 285 Val Asp Val Asn Asn Val Gly Ile Arg Gly Tyr Met Tyr Leu Lys Gly 290 295 300 Pro Arg Gly Ser Val Met Thr Thr Asn Ile Tyr Leu Asn Ser Ser Leu 305 310 315 320 Tyr Arg Gly Thr Lys Phe Ile Ile Lys Lys Tyr Ala Ser Gly Asn Lys 325 330 335 Asp Asn Ile Val Arg Asn Asn Asp Arg Val Tyr Ile Asn Val Val Val 340 345 350 Lys Asn Lys Glu Tyr Arg Leu Ala Thr Asn Ala Ser Gln Ala Gly Val 355 360 365 Glu Lys Ile Leu Ser Ala Leu Glu Ile Pro Asp Val Gly Asn Leu Ser 370 375 380 Gln Val Val Val Met Lys Ser Lys Asn Asp Gln Gly Ile Thr Asn Lys 385 390 395 400 Cys Lys Met Asn Leu Gln Asp Asn Asn Gly Asn Asp Ile Gly Phe Ile 405 410 415 Gly Phe His Gln Phe Asn Asn Ile Ala Lys Leu Val Ala Ser Asn Trp 420 425 430 Tyr Asn Arg Gln Ile Glu Arg Ser Ser Arg Thr Leu Gly Cys Ser Trp 435 440 445 Glu Phe Ile Pro Val Asp Asp Gly Trp Gly Glu Arg Pro Leu 450 455 460

27 ATG CAA TTT GTT AAT AAA CAA TTT AAT TAT AAA GAT CCT GTA AAT GGT 48 Met Gln Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn Gly 1 5 10 15 GTT GAT ATT GCT TAT ATA AAA ATT CCA AAT GTA GGA CAA ATG CAA CCA 96 Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Val Gly Gln Met Gln Pro 20 25 30 GTA AAA GCT TTT AAA ATT CAT AAT AAA ATA TGG GTT ATT CCA GAA AGA 144 Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro Glu Arg 35 40 45 GAT ACA TTT ACA AAT CCT GAA GAA GGA GAT TTA AAT CCA CCA CCA GAA 192 Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu 50 55 60 GCA AAA CAA GTT CCA GTT TCA TAT TAT GAT TCA ACA TAT TTA AGT ACA 240 Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr 65 70 75 80 GAT AAT GAA AAA GAT AAT TAT TTA AAG GGA GTT ACA AAA TTA TTT GAG 288 Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu 85 90 95 AGA ATT TAT TCA ACT GAT CTT GGA AGA ATG TTG TTA ACA TCA ATA GTA 336 Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile Val 100 105 110 AGG GGA ATA CCA TTT TGG GGT GGA AGT ACA ATA GAT ACA GAA TTA AAA 384 Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys 115 120 125 GTT ATT GAT ACT AAT TGT ATT AAT GTG ATA CAA CCA GAT GGT AGT TAT 432 Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly Ser Tyr 130 135 140 AGA TCA GAA GAA CTT AAT CTA GTA ATA ATA GGA CCC TCA GCT GAT ATT 480 Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile 145 150 155 160 ATA CAG TTT GAA TGT AAA AGC TTT GGA CAT GAA GTT TTG AAT CTT ACG 528 Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu Thr 165 170 175 CGA AAT GGT TAT GGC TCT ACT CAA TAC ATT AGA TTT AGC CCA GAT TTT 576 Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe 180 185 190 ACA TTT GGT TTT GAG GAG TCA CTT GAA GTT GAT ACA AAT CCT CTT TTA 624 Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu 195 200 205 GGT GCA GGC AAA TTT GCT ACA GAT CCA GCA GTA ACA TTA GCA CAT GAA 672 Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His Glu 210 215 220 CTT ATA CAT GCT GGA CAT AGA TTA TAT GGA ATA GCA ATT AAT CCA AAT 720 Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn 225 230 235 240 AGG GTT TTT AAA GTA AAT ACT AAT GCC TAT TAT GAA ATG AGT GGG TTA 768 Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu 245 250 255 GAA GTA AGC TTT GAG GAA CTT AGA ACA TTT GGG GGA CAT GAT GCA AAG 816 Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys 260 265 270 TTT ATA GAT AGT TTA CAG GAA AAC GAA TTT CGT CTA TAT TAT TAT AAT 864 Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn 275 280 285 AAG TTT AAA GAT ATA GCA AGT ACA CTT AAT AAA GCT AAA TCA ATA GTA 912 Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Val 290 295 300 GGT ACT ACT GCT TCA TTA CAG TAT ATG AAA AAT GTT TTT AAA GAG AAA 960 Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys Glu Lys 305 310 315 320 TAT CTC CTA TCT GAA GAT ACA TCT GGA AAA TTT TCG GTA GAT AAA TTA 1008 Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp Lys Leu 325 330 335 AAA TTT GAT AAG TTA TAC AAA ATG TTA ACA GAG ATT TAC ACA GAG GAT 1056 Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu Asp 340 345 350 AAT TTT GTT AAG TTT TTT AAA GTA CTT AAC AGA AAA ACA TAT TTG AAT 1104 Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn 355 360 365 TTT GAT AAA GCC GTA TTT AAG ATA AAT ATA GTA CCT AAG GTA AAT TAC 1152 Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn Tyr 370 375 380 ACA ATA TAT GAT GGA TTT AAT TTA AGA AAT ACA AAT TTA GCA GCA AAC 1200 Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn 385 390 395 400 TTT AAT GGT CAA AAT ACA GAA ATT AAT AAT ATG AAT TTT ACT AAA CTA 1248 Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu 405 410 415 AAA AAT TTT ACT GGA TTG TTT GAA TTT TAT AAG TTG CTA TGT GTA AGA 1296 Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg 420 425 430 GGG ATA ATA ACT TCT AAA ACT AAA TCA TTA GAT AAA GGA TAC AAT AAG 1344 Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Lys Gly Tyr Asn Lys 435 440 445 GCA TTA AAT GAT TTA TGT ATC AAA GTT AAT AAT TGG GAC TTG TTT TTT 1392 Ala Leu Asn Asp Leu Cys Ile Lys Val Asn Asn Trp Asp Leu Phe Phe 450 455 460 AGT CCT TCA GAA GAT AAT TTT ACT AAT GAT CTA AAT AAA GGA GAA GAA 1440 Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu Glu 465 470 475 480 ATT ACA TCT GAT ACT AAT ATA GAA GCA GCA GAA GAA AAT ATT AGT TTA 1488 Ile Thr Ser Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu 485 490 495 GAT TTA ATA CAA CAA TAT TAT TTA ACC TTT AAT TTT GAT AAT GAA CCT 1536 Asp Leu Ile Gln Gln Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro 500 505 510 GAA AAT ATT TCA ATA GAA AAT CTT TCA AGT GAC ATT ATA GGC CAA TTA 1584 Glu Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu 515 520 525 GAA CTT ATG CCT AAT ATA GAA AGA TTT CCT AAT GGA AAA AAG TAT GAG 1632 Glu Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu 530 535 540 TTA GAT AAA TAT ACT ATG TTC CAT TAT CTT CGT GCT CAA GAA TTT GAA 1680 Leu Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln Glu Phe Glu 545 550 555 560 CAT GGT AAA TCT AGG ATT GCT TTA ACA AAT TCT GTT AAC GAA GCA TTA 1728 His Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala Leu 565 570 575 TTA AAT CCT AGT CGT GTT TAT ACA TTT TTT TCT TCA GAC TAT GTA AAG 1776 Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys 580 585 590 AAA GTT AAT AAA GCT ACG GAG GCA GCT ATG TTT TTA GGC TGG GTA GAA 1824 Lys Val Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp Val Glu 595 600 605 CAA TTA GTA TAT GAT TTT ACC GAT GAA ACT AGC GAA GTA AGT ACT ACG 1872 Gln Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr 610 615 620 GAT AAA ATT GCG GAT ATA ACT ATA ATT ATT CCA TAT ATA GGA CCT GCT 1920 Asp Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala 625 630 635 640 TTA AAT ATA GGT AAT ATG TTA TAT AAA GAT GAT TTT GTA GGT GCT TTA 1968 Leu Asn Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu 645 650 655 ATA TTT TCA GGA GCT GTT ATT CTG TTA GAA TTT ATA CCA GAG ATT GCA 2016 Ile Phe Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala 660 665 670 ATA CCT GTA TTA GGT ACT TTT GCA CTT GTA TCA TAT ATT GCG AAT AAG 2064 Ile Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile Ala Asn Lys 675 680 685 GTT CTA ACC GTT CAA ACA ATA GAT AAT GCT TTA AGT AAA AGA AAT GAA 2112 Val Leu Thr Val Gln Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn Glu 690 695 700 AAA TGG GAT GAG GTC TAT AAA TAT ATA GTA ACA AAT TGG TTA GCA AAG 2160 Lys Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu Ala Lys 705 710 715 720 GTT AAT ACA CAG ATT GAT CTA ATA AGA AAA AAA ATG AAA GAA GCT TTA 2208 Val Asn Thr Gln Ile Asp Leu Ile Arg Lys Lys Met Lys Glu Ala Leu 725 730 735 GAA AAT CAA GCA GAA GCA ACA AAG GCT ATA ATA AAC TAT CAG TAT AAT 2256 Glu Asn Gln Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr Asn 740 745 750 CAA TAT ACT GAG GAA GAG AAA AAT AAT ATT AAT TTT AAT ATT GAT GAT 2304 Gln Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp 755 760 765 TTA AGT TCG AAA CTT AAT GAG TCT ATA AAT AAA GCT ATG ATT AAT ATA 2352 Leu Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn Ile 770 775 780 AAT AAA TTT TTG AAT CAA TGC TCT GTT TCA TAT TTA ATG AAT TCT ATG 2400 Asn Lys Phe Leu Asn Gln Cys Ser Val Ser Tyr Leu Met Asn Ser Met 785 790 795 800 ATC CCT TAT GGT GTT AAA CGG TTA GAA GAT TTT GAT GCT AGT CTT AAA 2448 Ile Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys 805 810 815 GAT GCA TTA TTA AAG TAT ATA TAT GAT AAT AGA GGA ACT TTA ATT GGT 2496 Asp Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Gly 820 825 830 CAA GTA GAT AGA TTA AAA GAT AAA GTT AAT AAT ACA CTT AGT ACA GAT 2544 Gln Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser Thr Asp 835 840 845 ATA CCT TTT CAG CTT TCC AAA TAC GTA GAT AAT CAA AGA TTA TTA TCT 2592 Ile Pro Phe Gln Leu Ser Lys Tyr Val Asp Asn Gln Arg Leu Leu Ser 850 855 860 ACA TTT ACT GAA TAT ATT AAG AAT ATT ATT AAT ACT TCT ATA TTG AAT 2640 Thr Phe Thr Glu Tyr Ile Lys Asn Ile Ile Asn Thr Ser Ile Leu Asn 865 870 875 880 TTA AGA TAT GAA AGT AAT CAT TTA ATA GAC TTA TCT AGG TAT GCA TCA 2688 Leu Arg Tyr Glu Ser Asn His Leu Ile Asp Leu Ser Arg Tyr Ala Ser 885 890 895 AAA ATA AAT ATT GGT AGT AAA GTA AAT TTT GAT CCA ATA GAT AAA AAT 2736 Lys Ile Asn Ile Gly Ser Lys Val Asn Phe Asp Pro Ile Asp Lys Asn 900 905 910 CAA ATT CAA TTA TTT AAT TTA GAA AGT AGT AAA ATT GAG GTA ATT TTA 2784 Gln Ile Gln Leu Phe Asn Leu Glu Ser Ser Lys Ile Glu Val Ile Leu 915 920 925 AAA AAT GCT ATT GTA TAT AAT AGT ATG TAT GAA AAT TTT AGT ACT AGC 2832 Lys Asn Ala Ile Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser 930 935 940 TTT TGG ATA AGA ATT CCT AAG TAT TTT AAC AGT ATA AGT CTA AAT AAT 2880 Phe Trp Ile Arg Ile Pro Lys Tyr Phe Asn Ser Ile Ser Leu Asn Asn 945 950 955 960 GAA TAT ACA ATA ATA AAT TGT ATG GAA AAT AAT TCA GGA TGG AAA GTA 2928 Glu Tyr Thr Ile Ile Asn Cys Met Glu Asn Asn Ser Gly Trp Lys Val 965 970 975 TCA CTT AAT TAT GGT GAA ATA ATC TGG ACT TTA CAG GAT ACT CAG GAA 2976 Ser Leu Asn Tyr Gly Glu Ile Ile Trp Thr Leu Gln Asp Thr Gln Glu 980 985 990 ATA AAA CAA AGA GTA GTT TTT AAA TAC AGT CAA ATG ATT AAT ATA TCA 3024 Ile Lys Gln Arg Val Val Phe Lys Tyr Ser Gln Met Ile Asn Ile Ser 995 1000 1005 GAT TAT ATA AAC AGA TGG ATT TTT GTA ACT ATC ACT AAT AAT AGA TTA 3072 Asp Tyr Ile Asn Arg Trp Ile Phe Val Thr Ile Thr Asn Asn Arg Leu 1010 1015 1020 AAT AAC TCT AAA ATT TAT ATA AAT GGA AGA TTA ATA GAT CAA AAA CCA 3120 Asn Asn Ser Lys Ile Tyr Ile Asn Gly Arg Leu Ile Asp Gln Lys Pro 1025 1030 1035 1040 ATT TCA AAT TTA GGT AAT ATT CAT GCT AGT AAT AAT ATA ATG TTT AAA 3168 Ile Ser Asn Leu Gly Asn Ile His Ala Ser Asn Asn Ile Met Phe Lys 1045 1050 1055 TTA GAT GGT TGT AGA GAT ACA CAT AGA TAT ATT TGG ATA AAA TAT TTT 3216 Leu Asp Gly Cys Arg Asp Thr His Arg Tyr Ile Trp Ile Lys Tyr Phe 1060 1065 1070 AAT CTT TTT GAT AAG GAA TTA AAT GAA AAA GAA ATC AAA GAT TTA TAT 3264 Asn Leu Phe Asp Lys Glu Leu Asn Glu Lys Glu Ile Lys Asp Leu Tyr 1075 1080 1085 GAT AAT CAA TCA AAT TCA GGT ATT TTA AAA GAC TTT TGG GGT GAT TAT 3312 Asp Asn Gln Ser Asn Ser Gly Ile Leu Lys Asp Phe Trp Gly Asp Tyr 1090 1095 1100 TTA CAA TAT GAT AAA CCA TAC TAT ATG TTA AAT TTA TAT GAT CCA AAT 3360 Leu Gln Tyr Asp Lys Pro Tyr Tyr Met Leu Asn Leu Tyr Asp Pro Asn 1105 1110 1115 1120 AAA TAT GTC GAT GTA AAT AAT GTA GGT ATT AGA GGT TAT ATG TAT CTT 3408 Lys Tyr Val Asp Val Asn Asn Val Gly Ile Arg Gly Tyr Met Tyr Leu 1125 1130 1135 AAA GGG CCT AGA GGT AGC GTA ATG ACT ACA AAC ATT TAT TTA AAT TCA 3456 Lys Gly Pro Arg Gly Ser Val Met Thr Thr Asn Ile Tyr Leu Asn Ser 1140 1145 1150 AGT TTG TAT AGG GGG ACA AAA TTT ATT ATA AAA AAA TAT GCT TCT GGA 3504 Ser Leu Tyr Arg Gly Thr Lys Phe Ile Ile Lys Lys Tyr Ala Ser Gly 1155 1160 1165 AAT AAA GAT AAT ATT GTT AGA AAT AAT GAT CGT GTA TAT ATT AAT GTA 3552 Asn Lys Asp Asn Ile Val Arg Asn Asn Asp Arg Val Tyr Ile Asn Val 1170 1175 1180 GTA GTT AAA AAT AAA GAA TAT AGG TTA GCT ACT AAT GCA TCA CAG GCA 3600 Val Val Lys Asn Lys Glu Tyr Arg Leu Ala Thr Asn Ala Ser Gln Ala 1185 1190 1195 1200 GGC GTA GAA AAA ATA CTA AGT GCA TTA GAA ATA CCT GAT GTA GGA AAT 3648 Gly Val Glu Lys Ile Leu Ser Ala Leu Glu Ile Pro Asp Val Gly Asn 1205 1210 1215 CTA AGT CAA GTA GTA GTA ATG AAG TCA AAA AAT GAT CAA GGA ATA ACA 3696 Leu Ser Gln Val Val Val Met Lys Ser Lys Asn Asp Gln Gly Ile Thr 1220 1225 1230 AAT AAA TGC AAA ATG AAT TTA CAA GAT AAT AAT GGG AAT GAT ATA GGC 3744 Asn Lys Cys Lys Met Asn Leu Gln Asp Asn Asn Gly Asn Asp Ile Gly 1235 1240 1245 TTT ATA GGA TTT CAT CAG TTT AAT AAT ATA GCT AAA CTA GTA GCA AGT 3792 Phe Ile Gly Phe His Gln Phe Asn Asn Ile Ala Lys Leu Val Ala Ser 1250 1255 1260 AAT TGG TAT AAT AGA CAA ATA GAA AGA TCT AGT AGG ACT TTG GGT TGC 3840 Asn Trp Tyr Asn Arg Gln Ile Glu Arg Ser Ser Arg Thr Leu Gly Cys 1265 1270 1275 1280 TCA TGG GAA TTT ATT CCT GTA GAT GAT GGA TGG GGA GAA AGG CCA CTG 3888 Ser Trp Glu Phe Ile Pro Val Asp Asp Gly Trp Gly Glu Arg Pro Leu 1285 1290 1295 TAA 3891

28 Met Gln Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn Gly 1 5 10 15 Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Val Gly Gln Met Gln Pro 20 25 30 Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro Glu Arg 35 40 45 Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu 50 55 60 Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr 65 70 75 80 Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu 85 90 95 Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile Val 100 105 110 Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys 115 120 125 Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly Ser Tyr 130 135 140 Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile 145 150 155 160 Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu Thr 165 170 175 Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe 180 185 190 Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu 195 200 205 Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His Glu 210 215 220 Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn 225 230 235 240 Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu 245 250 255 Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys 260 265 270 Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn 275 280 285 Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Val 290 295 300 Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys Glu Lys 305 310 315 320 Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp Lys Leu 325 330 335 Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu Asp 340 345 350 Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn 355 360 365 Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn Tyr 370 375 380 Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn 385 390 395 400 Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu 405 410 415 Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg 420 425 430 Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Lys Gly Tyr Asn Lys 435 440 445 Ala Leu Asn Asp Leu Cys Ile Lys Val Asn Asn Trp Asp Leu Phe Phe 450 455 460 Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu Glu 465 470 475 480 Ile Thr Ser Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu 485 490 495 Asp Leu Ile Gln Gln Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro 500 505 510 Glu Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu 515 520 525 Glu Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu 530 535 540 Leu Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln Glu Phe Glu 545 550 555 560 His Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala Leu 565 570 575 Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys 580 585 590 Lys Val Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp Val Glu 595 600 605 Gln Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr 610 615 620 Asp Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala 625 630 635 640 Leu Asn Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu 645 650 655 Ile Phe Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala 660 665 670 Ile Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile Ala Asn Lys 675 680 685 Val Leu Thr Val Gln Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn Glu 690 695 700 Lys Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu Ala Lys 705 710 715 720 Val Asn Thr Gln Ile Asp Leu Ile Arg Lys Lys Met Lys Glu Ala Leu 725 730 735 Glu Asn Gln Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr Asn 740 745 750 Gln Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp 755 760 765 Leu Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn Ile 770 775 780 Asn Lys Phe Leu Asn Gln Cys Ser Val Ser Tyr Leu Met Asn Ser Met 785 790 795 800 Ile Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys 805 810 815 Asp Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Gly 820 825 830 Gln Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser Thr Asp 835 840 845 Ile Pro Phe Gln Leu Ser Lys Tyr Val Asp Asn Gln Arg Leu Leu Ser 850 855 860 Thr Phe Thr Glu Tyr Ile Lys Asn Ile Ile Asn Thr Ser Ile Leu Asn 865 870 875 880 Leu Arg Tyr Glu Ser Asn His Leu Ile Asp Leu Ser Arg Tyr Ala Ser 885 890 895 Lys Ile Asn Ile Gly Ser Lys Val Asn Phe Asp Pro Ile Asp Lys Asn 900 905 910 Gln Ile Gln Leu Phe Asn Leu Glu Ser Ser Lys Ile Glu Val Ile Leu 915 920 925 Lys Asn Ala Ile Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser 930 935 940 Phe Trp Ile Arg Ile Pro Lys Tyr Phe Asn Ser Ile Ser Leu Asn Asn 945 950 955 960 Glu Tyr Thr Ile Ile Asn Cys Met Glu Asn Asn Ser Gly Trp Lys Val 965 970 975 Ser Leu Asn Tyr Gly Glu Ile Ile Trp Thr Leu Gln Asp Thr Gln Glu 980 985 990 Ile Lys Gln Arg Val Val Phe Lys Tyr Ser Gln Met Ile Asn Ile Ser 995 1000 1005 Asp Tyr Ile Asn Arg Trp Ile Phe Val Thr Ile Thr Asn Asn Arg Leu 1010 1015 1020 Asn Asn Ser Lys Ile Tyr Ile Asn Gly Arg Leu Ile Asp Gln Lys Pro 1025 1030 1035 1040 Ile Ser Asn Leu Gly Asn Ile His Ala Ser Asn Asn Ile Met Phe Lys 1045 1050 1055 Leu Asp Gly Cys Arg Asp Thr His Arg Tyr Ile Trp Ile Lys Tyr Phe 1060 1065 1070 Asn Leu Phe Asp Lys Glu Leu Asn Glu Lys Glu Ile Lys Asp Leu Tyr 1075 1080 1085 Asp Asn Gln Ser Asn Ser Gly Ile Leu Lys Asp Phe Trp Gly Asp Tyr 1090 1095 1100 Leu Gln Tyr Asp Lys Pro Tyr Tyr Met Leu Asn Leu Tyr Asp Pro Asn 1105 1110 1115 1120 Lys Tyr Val Asp Val Asn Asn Val Gly Ile Arg Gly Tyr Met Tyr Leu 1125 1130 1135 Lys Gly Pro Arg Gly Ser Val Met Thr Thr Asn Ile Tyr Leu Asn Ser 1140 1145 1150 Ser Leu Tyr Arg Gly Thr Lys Phe Ile Ile Lys Lys Tyr Ala Ser Gly 1155 1160 1165 Asn Lys Asp Asn Ile Val Arg Asn Asn Asp Arg Val Tyr Ile Asn Val 1170 1175 1180 Val Val Lys Asn Lys Glu Tyr Arg Leu Ala Thr Asn Ala Ser Gln Ala 1185 1190 1195 1200 Gly Val Glu Lys Ile Leu Ser Ala Leu Glu Ile Pro Asp Val Gly Asn 1205 1210 1215 Leu Ser Gln Val Val Val Met Lys Ser Lys Asn Asp Gln Gly Ile Thr 1220 1225 1230 Asn Lys Cys Lys Met Asn Leu Gln Asp Asn Asn Gly Asn Asp Ile Gly 1235 1240 1245 Phe Ile Gly Phe His Gln Phe Asn Asn Ile Ala Lys Leu Val Ala Ser 1250 1255 1260 Asn Trp Tyr Asn Arg Gln Ile Glu Arg Ser Ser Arg Thr Leu Gly Cys 1265 1270 1275 1280 Ser Trp Glu Phe Ile Pro Val Asp Asp Gly Trp Gly Glu Arg Pro Leu 1285 1290 1295

29 CGCCATGGCT AGATTATTAT CTACATTTAC 30

30 GCAAGCTTCT TGACAGACTC ATGTAG 26

31 AGATCTCGAT CCCGCGAAAT TAATACGACT CACTATAGGG GAATTGTGAG CGGATAACAA 60 TTCCCCTCTA GAAATAATTT TGTTTAACTT TAAGAAGGAG ATATACCATG GGCCATCATC 120 ATCATCATCA TCATCATCAT CACAGCAGCG GCCATATCGA AGGTCGTCAT ATGGCTAGCA 180 TGGCTAGATT ATTATCTACA TTTACTGAAT ATATTAAGAA TATTATTAAT ACTTCTATAT 240 TGAATTTAAG ATATGAAAGT AATCATTTAA TAGACTTATC TAGGTATGCA TCAAAAATAA 300 ATATTGGTAG TAAAGTAAAT TTTGATCCAA TAGATAAAAA TCAAATTCAA TTATTTAATT 360 TAGAAAGTAG TAAAATTGAG GTAATTTTAA AAAATGCTAT TGTATATAAT AGTATGTATG 420 AAAATTTTAG TACTAGCTTT TGGATAAGAA TTCCTAAGTA TTTTAACAGT ATAAGTCTAA 480 ATAATGAATA TACAATAATA AATTGTATGG AAAATAATTC AGGATGGAAA GTATCACTTA 540 ATTATGGTGA AATAATCTGG ACTTTACAGG ATACTCAGGA AATAAAACAA AGAGTAGTTT 600 TTAAATACAG TCAAATGATT AATATATCAG ATTATATAAA CAGATGGATT TTTGTAACTA 660 TCACTAATAA TAGATTAAAT AACTCTAAAA TTTATATAAA TGGAAGATTA ATAGATCAAA 720 AACCAATTTC AAATTTAGGT AATATTCATG CTAGTAATAA TATAATGTTT AAATTAGATG 780 GTTGTAGAGA TACACATAGA TATATTTGGA TAAAATATTT TAATCTTTTT GATAAGGAAT 840 TAAATGAAAA AGAAATCAAA GATTTATATG ATAATCAATC AAATTCAGGT ATTTTAAAAG 900 ACTTTTGGGG TGATTATTTA CAATATGATA AACCATACTA TATGTTAAAT TTATATGATC 960 CAAATAAATA TGTCGATGTA AATAATGTAG GTATTAGAGG TTATATGTAT CTTAAAGGGC 1020 CTAGAGGTAG CGTAATGACT ACAAACATTT ATTTAAATTC AAGTTTGTAT AGGGGGACAA 1080 AATTTATTAT AAAAAAATAT GCTTCTGGAA ATAAAGATAA TATTGTTAGA AATAATGATC 1140 GTGTATATAT TAATGTAGTA GTTAAAAATA AAGAATATAG GTTAGCTACT AATGCATCAC 1200 AGGCAGGCGT AGAAAAAATA CTAAGTGCAT TAGAAATACC TGATGTAGGA AATCTAAGTC 1260 AAGTAGTAGT AATGAAGTCA AAAAATGATC AAGGAATAAC AAATAAATGC AAAATGAATT 1320 TACAAGATAA TAATGGGAAT GATATAGGCT TTATAGGATT TCATCAGTTT AATAATATAG 1380 CTAAACTAGT AGCAAGTAAT TGGTATAATA GACAAATAGA AAGATCTAGT AGGACTTTGG 1440 GTTGCTCATG GGAATTTATT CCTGTAGATG ATGGATGGGG AGAAAGGCCA CTGTAATTAA 1500 TCTCAAACTA CATGAGTCTG TCAAGAAGCT TGCGGCCGCA CTCGAG 1546

32 Met His His His His His His Met Ala 1 5

33 TATGCATCAC CATCACCATC A 21

34 CATGTGATGG TGATGGTGAT GCA 23

35 ATG CAT CAC CAT CAC CAT CAC ATG GCT CGT CTG CTG TCT ACC TTC ACT 48 Met His His His His His His Met Ala Arg Leu Leu Ser Thr Phe Thr 1 5 10 15 GAA TAC ATC AAG AAC ATC ATC AAT ACC TCC ATC CTG AAC CTG CGC TAC 96 Glu Tyr Ile Lys Asn Ile Ile Asn Thr Ser Ile Leu Asn Leu Arg Tyr 20 25 30 GAA TCC AAT CAC CTG ATC GAC CTG TCT CGC TAC GCT TCC AAA ATC AAC 144 Glu Ser Asn His Leu Ile Asp Leu Ser Arg Tyr Ala Ser Lys Ile Asn 35 40 45 ATC GGT TCT AAA GTT AAC TTC GAT CCG ATC GAC AAG AAT CAG ATC CAG 192 Ile Gly Ser Lys Val Asn Phe Asp Pro Ile Asp Lys Asn Gln Ile Gln 50 55 60 CTG TTC AAT CTG GAA TCT TCC AAA ATC GAA GTT ATC CTG AAG AAT GCT 240 Leu Phe Asn Leu Glu Ser Ser Lys Ile Glu Val Ile Leu Lys Asn Ala 65 70 75 80 ATC GTA TAC AAC TCT ATG TAC GAA AAC TTC TCC ACC TCC TTC TGG ATC 288 Ile Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser Phe Trp Ile 85 90 95 CGT ATC CCG AAA TAC TTC AAC TCC ATC TCT CTG AAC AAT GAA TAC ACC 336 Arg Ile Pro Lys Tyr Phe Asn Ser Ile Ser Leu Asn Asn Glu Tyr Thr 100 105 110 ATC ATC AAC TGC ATG GAA AAC AAT TCT GGT TGG AAA GTA TCT CTG AAC 384 Ile Ile Asn Cys Met Glu Asn Asn Ser Gly Trp Lys Val Ser Leu Asn 115 120 125 TAC GGT GAA ATC ATC TGG ACT CTG CAG GAC ACT CAG GAA ATC AAA CAG 432 Tyr Gly Glu Ile Ile Trp Thr Leu Gln Asp Thr Gln Glu Ile Lys Gln 130 135 140 CGT GTT GTA TTC AAA TAC TCT CAG ATG ATC AAC ATC TCT GAC TAC ATC 480 Arg Val Val Phe Lys Tyr Ser Gln Met Ile Asn Ile Ser Asp Tyr Ile 145 150 155 160 AAT CGC TGG ATC TTC GTT ACC ATC ACC AAC AAT CGT CTG AAT AAC TCC 528 Asn Arg Trp Ile Phe Val Thr Ile Thr Asn Asn Arg Leu Asn Asn Ser 165 170 175 AAA ATC TAC ATC AAC GGC CGT CTG ATC GAC CAG AAA CCG ATC TCC AAT 576 Lys Ile Tyr Ile Asn Gly Arg Leu Ile Asp Gln Lys Pro Ile Ser Asn 180 185 190 CTG GGT AAC ATC CAC GCT TCT AAT AAC ATC ATG TTC AAA CTG GAC GGT 624 Leu Gly Asn Ile His Ala Ser Asn Asn Ile Met Phe Lys Leu Asp Gly 195 200 205 TGT CGT GAC ACT CAC CGC TAC ATC TGG ATC AAA TAC TTC AAT CTG TTC 672 Cys Arg Asp Thr His Arg Tyr Ile Trp Ile Lys Tyr Phe Asn Leu Phe 210 215 220 GAC AAA GAA CTG AAC GAA AAA GAA ATC AAA GAC CTG TAC GAC AAC CAG 720 Asp Lys Glu Leu Asn Glu Lys Glu Ile Lys Asp Leu Tyr Asp Asn Gln 225 230 235 240 TCC AAT TCT GGT ATC CTG AAA GAC TTC TGG GGT GAC TAC CTG CAG TAC 768 Ser Asn Ser Gly Ile Leu Lys Asp Phe Trp Gly Asp Tyr Leu Gln Tyr 245 250 255 GAC AAA CCG TAC TAC ATG CTG AAT CTG TAC GAT CCG AAC AAA TAC GTT 816 Asp Lys Pro Tyr Tyr Met Leu Asn Leu Tyr Asp Pro Asn Lys Tyr Val 260 265 270 GAC GTC AAC AAT GTA GGT ATC CGC GGT TAC ATG TAC CTG AAA GGT CCG 864 Asp Val Asn Asn Val Gly Ile Arg Gly Tyr Met Tyr Leu Lys Gly Pro 275 280 285 CGT GGT TCT GTT ATG ACT ACC AAC ATC TAC CTG AAC TCT TCC CTG TAC 912 Arg Gly Ser Val Met Thr Thr Asn Ile Tyr Leu Asn Ser Ser Leu Tyr 290 295 300 CGT GGT ACC AAA TTC ATC ATC AAG AAA TAC GCG TCT GGT AAC AAG GAC 960 Arg Gly Thr Lys Phe Ile Ile Lys Lys Tyr Ala Ser Gly Asn Lys Asp 305 310 315 320 AAT ATC GTT CGC AAC AAT GAT CGT GTA TAC ATC AAT GTT GTA GTT AAG 1008 Asn Ile Val Arg Asn Asn Asp Arg Val Tyr Ile Asn Val Val Val Lys 325 330 335 AAC AAA GAA TAC CGT CTG GCT ACC AAT GCT TCT CAG GCT GGT GTA GAA 1056 Asn Lys Glu Tyr Arg Leu Ala Thr Asn Ala Ser Gln Ala Gly Val Glu 340 345 350 AAG ATC TTG TCT GCT CTG GAA ATC CCG GAC GTT GGT AAT CTG TCT CAG 1104 Lys Ile Leu Ser Ala Leu Glu Ile Pro Asp Val Gly Asn Leu Ser Gln 355 360 365 GTA GTT GTA ATG AAA TCC AAG AAC GAC CAG GGT ATC ACT AAC AAA TGC 1152 Val Val Val Met Lys Ser Lys Asn Asp Gln Gly Ile Thr Asn Lys Cys 370 375 380 AAA ATG AAT CTG CAG GAC AAC AAT GGT AAC GAT ATC GGT TTC ATC GGT 1200 Lys Met Asn Leu Gln Asp Asn Asn Gly Asn Asp Ile Gly Phe Ile Gly 385 390 395 400 TTC CAC CAG TTC AAC AAT ATC GCT AAA CTG GTT GCT TCC AAC TGG TAC 1248 Phe His Gln Phe Asn Asn Ile Ala Lys Leu Val Ala Ser Asn Trp Tyr 405 410 415 AAT CGT CAG ATC GAA CGT TCC TCT CGC ACT CTG GGT TGC TCT TGG GAG 1296 Asn Arg Gln Ile Glu Arg Ser Ser Arg Thr Leu Gly Cys Ser Trp Glu 420 425 430 TTC ATC CCG GTT GAT GAC GGT TGG GGT GAA CGT CCG CTG TAACCCGGGA 1345 Phe Ile Pro Val Asp Asp Gly Trp Gly Glu Arg Pro Leu 435 440 445 AAGCTT 1351

36 Met His His His His His His Met Ala Arg Leu Leu Ser Thr Phe Thr 1 5 10 15 Glu Tyr Ile Lys Asn Ile Ile Asn Thr Ser Ile Leu Asn Leu Arg Tyr 20 25 30 Glu Ser Asn His Leu Ile Asp Leu Ser Arg Tyr Ala Ser Lys Ile Asn 35 40 45 Ile Gly Ser Lys Val Asn Phe Asp Pro Ile Asp Lys Asn Gln Ile Gln 50 55 60 Leu Phe Asn Leu Glu Ser Ser Lys Ile Glu Val Ile Leu Lys Asn Ala 65 70 75 80 Ile Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser Phe Trp Ile 85 90 95 Arg Ile Pro Lys Tyr Phe Asn Ser Ile Ser Leu Asn Asn Glu Tyr Thr 100 105 110 Ile Ile Asn Cys Met Glu Asn Asn Ser Gly Trp Lys Val Ser Leu Asn 115 120 125 Tyr Gly Glu Ile Ile Trp Thr Leu Gln Asp Thr Gln Glu Ile Lys Gln 130 135 140 Arg Val Val Phe Lys Tyr Ser Gln Met Ile Asn Ile Ser Asp Tyr Ile 145 150 155 160 Asn Arg Trp Ile Phe Val Thr Ile Thr Asn Asn Arg Leu Asn Asn Ser 165 170 175 Lys Ile Tyr Ile Asn Gly Arg Leu Ile Asp Gln Lys Pro Ile Ser Asn 180 185 190 Leu Gly Asn Ile His Ala Ser Asn Asn Ile Met Phe Lys Leu Asp Gly 195 200 205 Cys Arg Asp Thr His Arg Tyr Ile Trp Ile Lys Tyr Phe Asn Leu Phe 210 215 220 Asp Lys Glu Leu Asn Glu Lys Glu Ile Lys Asp Leu Tyr Asp Asn Gln 225 230 235 240 Ser Asn Ser Gly Ile Leu Lys Asp Phe Trp Gly Asp Tyr Leu Gln Tyr 245 250 255 Asp Lys Pro Tyr Tyr Met Leu Asn Leu Tyr Asp Pro Asn Lys Tyr Val 260 265 270 Asp Val Asn Asn Val Gly Ile Arg Gly Tyr Met Tyr Leu Lys Gly Pro 275 280 285 Arg Gly Ser Val Met Thr Thr Asn Ile Tyr Leu Asn Ser Ser Leu Tyr 290 295 300 Arg Gly Thr Lys Phe Ile Ile Lys Lys Tyr Ala Ser Gly Asn Lys Asp 305 310 315 320 Asn Ile Val Arg Asn Asn Asp Arg Val Tyr Ile Asn Val Val Val Lys 325 330 335 Asn Lys Glu Tyr Arg Leu Ala Thr Asn Ala Ser Gln Ala Gly Val Glu 340 345 350 Lys Ile Leu Ser Ala Leu Glu Ile Pro Asp Val Gly Asn Leu Ser Gln 355 360 365 Val Val Val Met Lys Ser Lys Asn Asp Gln Gly Ile Thr Asn Lys Cys 370 375 380 Lys Met Asn Leu Gln Asp Asn Asn Gly Asn Asp Ile Gly Phe Ile Gly 385 390 395 400 Phe His Gln Phe Asn Asn Ile Ala Lys Leu Val Ala Ser Asn Trp Tyr 405 410 415 Asn Arg Gln Ile Glu Arg Ser Ser Arg Thr Leu Gly Cys Ser Trp Glu 420 425 430 Phe Ile Pro Val Asp Asp Gly Trp Gly Glu Arg Pro Leu 435 440 445

37 CGCATATGAA TATTCGTCCA TTGCATG 27

38 GGAAGCTTGC AGGGCAATTA CATCATG 27

39 ATG CCA GTT ACA ATA AAT AAT TTT AAT TAT AAT GAT CCT ATT GAT AAT 48 Met Pro Val Thr Ile Asn Asn Phe Asn Tyr Asn Asp Pro Ile Asp Asn 1 5 10 15 GAC AAT ATT ATT ATG ATG GAA CCT CCA TTT GCA AGG GGT ACG GGG AGA 96 Asp Asn Ile Ile Met Met Glu Pro Pro Phe Ala Arg Gly Thr Gly Arg 20 25 30 TAT TAT AAA GCT TTT AAA ATC ACA GAT CGT ATT TGG ATA ATA CCC GAA 144 Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Ile Pro Glu 35 40 45 AGA TAT ACT TTT GGA TAT AAA CCT GAG GAT TTT AAT AAA AGT TCC GGT 192 Arg Tyr Thr Phe Gly Tyr Lys Pro Glu Asp Phe Asn Lys Ser Ser Gly 50 55 60 ATT TTT AAT AGA GAT GTT TGT GAA TAT TAT GAT CCA GAT TAC TTA AAT 240 Ile Phe Asn Arg Asp Val Cys Glu Tyr Tyr Asp Pro Asp Tyr Leu Asn 65 70 75 80 ACC AAT GAT AAA AAG AAT ATA TTT TTC CAA ACA TTG ATC AAG TTA TTT 288 Thr Asn Asp Lys Lys Asn Ile Phe Phe Gln Thr Leu Ile Lys Leu Phe 85 90 95 AAT AGA ATC AAA TCA AAA CCA TTG GGT GAA AAG TTA TTA GAG ATG ATT 336 Asn Arg Ile Lys Ser Lys Pro Leu Gly Glu Lys Leu Leu Glu Met Ile 100 105 110 ATA AAT GGT ATA CCT TAT CTT GGA GAT AGA CGT GTT CCA CTC GAA GAG 384 Ile Asn Gly Ile Pro Tyr Leu Gly Asp Arg Arg Val Pro Leu Glu Glu 115 120 125 TTT AAC ACA AAC ATT GCT AGT GTA ACT GTT AAT AAA TTA ATT AGT AAT 432 Phe Asn Thr Asn Ile Ala Ser Val Thr Val Asn Lys Leu Ile Ser Asn 130 135 140 CCA GGA GAA GTG GAG CGA AAA AAA GGT ATT TTC GCA AAT TTA ATA ATA 480 Pro Gly Glu Val Glu Arg Lys Lys Gly Ile Phe Ala Asn Leu Ile Ile 145 150 155 160 TTT GGA CCT GGG CCA GTT TTA AAT GAA AAT GAG ACT ATA GAT ATA GGT 528 Phe Gly Pro Gly Pro Val Leu Asn Glu Asn Glu Thr Ile Asp Ile Gly 165 170 175 ATA CAA AAT CAT TTT GCA TCA AGG GAA GGC TTT GGG GGT ATA ATG CAA 576 Ile Gln Asn His Phe Ala Ser Arg Glu Gly Phe Gly Gly Ile Met Gln 180 185 190 ATG AAA TTT TGT CCA GAA TAT GTA AGC GTA TTT AAT AAT GTT CAA GAA 624 Met Lys Phe Cys Pro Glu Tyr Val Ser Val Phe Asn Asn Val Gln Glu 195 200 205 AAC AAA GGC GCA AGT ATA TTT AAT AGA CGT GGA TAT TTT TCA GAT CCA 672 Asn Lys Gly Ala Ser Ile Phe Asn Arg Arg Gly Tyr Phe Ser Asp Pro 210 215 220 GCC TTG ATA TTA ATG CAT GAA CTT ATA CAT GTT TTG CAT GGA TTA TAT 720 Ala Leu Ile Leu Met His Glu Leu Ile His Val Leu His Gly Leu Tyr 225 230 235 240 GGC ATT AAA GTA GAT GAT TTA CCA ATT GTA CCA AAT GAA AAA AAA TTT 768 Gly Ile Lys Val Asp Asp Leu Pro Ile Val Pro Asn Glu Lys Lys Phe 245 250 255 TTT ATG CAA TCT ACA GAT ACT ATA CAG GCA GAA GAA CTA TAT ACA TTT 816 Phe Met Gln Ser Thr Asp Thr Ile Gln Ala Glu Glu Leu Tyr Thr Phe 260 265 270 GGA GGA CAA GAT CCC AGC ATC ATA TCT CCT TCT ACA GAT AAA AGT ATC 864 Gly Gly Gln Asp Pro Ser Ile Ile Ser Pro Ser Thr Asp Lys Ser Ile 275 280 285 TAT GAT AAA GTT TTG CAA AAT TTT AGG GGG ATA GTT GAT AGA CTT AAC 912 Tyr Asp Lys Val Leu Gln Asn Phe Arg Gly Ile Val Asp Arg Leu Asn 290 295 300 AAG GTT TTA GTT TGC ATA TCA GAT CCT AAC ATT AAC ATT AAT ATA TAT 960 Lys Val Leu Val Cys Ile Ser Asp Pro Asn Ile Asn Ile Asn Ile Tyr 305 310 315 320 AAA AAT AAA TTT AAA GAT AAA TAT AAA TTC GTT GAA GAT TCT GAA GGA 1008 Lys Asn Lys Phe Lys Asp Lys Tyr Lys Phe Val Glu Asp Ser Glu Gly 325 330 335 AAA TAT AGT ATA GAT GTA GAA AGT TTC AAT AAA TTA TAT AAA AGC TTA 1056 Lys Tyr Ser Ile Asp Val Glu Ser Phe Asn Lys Leu Tyr Lys Ser Leu 340 345 350 ATG TTA GGT TTT ACA GAA ATT AAT ATA GCA GAA AAT TAT AAA ATA AAA 1104 Met Leu Gly Phe Thr Glu Ile Asn Ile Ala Glu Asn Tyr Lys Ile Lys 355 360 365 ACT AGA GCT TCT TAT TTT AGT GAT TCC TTA CCA CCA GTA AAA ATA AAA 1152 Thr Arg Ala Ser Tyr Phe Ser Asp Ser Leu Pro Pro Val Lys Ile Lys 370 375 380 AAT TTA TTA GAT AAT GAA ATC TAT ACT ATA GAG GAA GGG TTT AAT ATA 1200 Asn Leu Leu Asp Asn Glu Ile Tyr Thr Ile Glu Glu Gly Phe Asn Ile 385 390 395 400 TCT GAT AAA AAT ATG GGA AAA GAA TAT AGG GGT CAG AAT AAA GCT ATA 1248 Ser Asp Lys Asn Met Gly Lys Glu Tyr Arg Gly Gln Asn Lys Ala Ile 405 410 415 AAT AAA CAA GCT TAT GAA GAA ATC AGC AAG GAG CAT TTG GCT GTA TAT 1296 Asn Lys Gln Ala Tyr Glu Glu Ile Ser Lys Glu His Leu Ala Val Tyr 420 425 430 AAG ATA CAA ATG TGT AAA AGT GTT AAA GTT CCA GGA ATA TGT ATT GAT 1344 Lys Ile Gln Met Cys Lys Ser Val Lys Val Pro Gly Ile Cys Ile Asp 435 440 445 GTC GAT AAT GAA AAT TTG TTC TTT ATA GCT GAT AAA AAT AGT TTT TCA 1392 Val Asp Asn Glu Asn Leu Phe Phe Ile Ala Asp Lys Asn Ser Phe Ser 450 455 460 GAT GAT TTA TCT AAA AAT GAA AGA GTA GAA TAT AAT ACA CAG AAT AAT 1440 Asp Asp Leu Ser Lys Asn Glu Arg Val Glu Tyr Asn Thr Gln Asn Asn 465 470 475 480 TAT ATA GGA AAT GAC TTT CCT ATA AAT GAA TTA ATT TTA GAT ACT GAT 1488 Tyr Ile Gly Asn Asp Phe Pro Ile Asn Glu Leu Ile Leu Asp Thr Asp 485 490 495 TTA ATA AGT AAA ATA GAA TTA CCA AGT GAA AAT ACA GAA TCA CTT ACT 1536 Leu Ile Ser Lys Ile Glu Leu Pro Ser Glu Asn Thr Glu Ser Leu Thr 500 505 510 GAT TTT AAT GTA GAT GTT CCA GTA TAT GAA AAA CAA CCC GCT ATA AAA 1584 Asp Phe Asn Val Asp Val Pro Val Tyr Glu Lys Gln Pro Ala Ile Lys 515 520 525 AAA GTT TTT ACA GAT GAA AAT ACC ATC TTT CAA TAT TTA TAC TCT CAG 1632 Lys Val Phe Thr Asp Glu Asn Thr Ile Phe Gln Tyr Leu Tyr Ser Gln 530 535 540 ACA TTT CCT CTA AAT ATA AGA GAT ATA AGT TTA ACA TCT TCA TTT GAT 1680 Thr Phe Pro Leu Asn Ile Arg Asp Ile Ser Leu Thr Ser Ser Phe Asp 545 550 555 560 GAT GCA TTA TTA GTT TCT AGC AAA GTT TAT TCA TTT TTT TCT ATG GAT 1728 Asp Ala Leu Leu Val Ser Ser Lys Val Tyr Ser Phe Phe Ser Met Asp 565 570 575 TAT ATT AAA ACT GCT AAT AAA GTA GTA GAA GCA GGA TTA TTT GCA GGT 1776 Tyr Ile Lys Thr Ala Asn Lys Val Val Glu Ala Gly Leu Phe Ala Gly 580 585 590 TGG GTG AAA CAG ATA GTA GAT GAT TTT GTA ATC GAA GCT AAT AAA AGC 1824 Trp Val Lys Gln Ile Val Asp Asp Phe Val Ile Glu Ala Asn Lys Ser 595 600 605 AGT ACT ATG GAT AAA ATT GCA GAT ATA TCT CTA ATT GTT CCT TAT ATA 1872 Ser Thr Met Asp Lys Ile Ala Asp Ile Ser Leu Ile Val Pro Tyr Ile 610 615 620 GGA TTA GCT TTA AAT GTA GGA GAT GAA ACA GCT AAA GGA AAT TTT GAA 1920 Gly Leu Ala Leu Asn Val Gly Asp Glu Thr Ala Lys Gly Asn Phe Glu 625 630 635 640 AGT GCT TTT GAG ATT GCA GGA TCC AGT ATT TTA CTA GAA TTT ATA CCA 1968 Ser Ala Phe Glu Ile Ala Gly Ser Ser Ile Leu Leu Glu Phe Ile Pro 645 650 655 GAA CTT TTA ATA CCT GTA GTT GGA GTC TTT TTA TTA GAA TCA TAT ATT 2016 Glu Leu Leu Ile Pro Val Val Gly Val Phe Leu Leu Glu Ser Tyr Ile 660 665 670 GAC AAT AAA AAT AAA ATT ATT AAA ACA ATA GAT AAT GCT TTA ACT AAA 2064 Asp Asn Lys Asn Lys Ile Ile Lys Thr Ile Asp Asn Ala Leu Thr Lys 675 680 685 AGA GTG GAA AAA TGG ATT GAT ATG TAC GGA TTA ATA GTA GCG CAA TGG 2112 Arg Val Glu Lys Trp Ile Asp Met Tyr Gly Leu Ile Val Ala Gln Trp 690 695 700 CTC TCA ACA GTT AAT ACT CAA TTT TAT ACA ATA AAA GAG GGA ATG TAT 2160 Leu Ser Thr Val Asn Thr Gln Phe Tyr Thr Ile Lys Glu Gly Met Tyr 705 710 715 720 AAG GCT TTA AAT TAT CAA GCA CAA GCA TTG GAA GAA ATA ATA AAA TAC 2208 Lys Ala Leu Asn Tyr Gln Ala Gln Ala Leu Glu Glu Ile Ile Lys Tyr 725 730 735 AAA TAT AAT ATA TAT TCT GAA GAG GAA AAG TCA AAT ATT AAC ATC AAT 2256 Lys Tyr Asn Ile Tyr Ser Glu Glu Glu Lys Ser Asn Ile Asn Ile Asn 740 745 750 TTT AAT GAT ATA AAT TCT AAA CTT AAT GAT GGT ATT AAC CAA GCT ATG 2304 Phe Asn Asp Ile Asn Ser Lys Leu Asn Asp Gly Ile Asn Gln Ala Met 755 760 765 GAT AAT ATA AAT GAT TTT ATA AAT GAA TGT TCT GTA TCA TAT TTA ATG 2352 Asp Asn Ile Asn Asp Phe Ile Asn Glu Cys Ser Val Ser Tyr Leu Met 770 775 780 AAA AAA ATG ATT CCA TTA GCT GTA AAA AAA TTA CTA GAC TTT GAT AAT 2400 Lys Lys Met Ile Pro Leu Ala Val Lys Lys Leu Leu Asp Phe Asp Asn 785 790 795 800 ACT CTC AAA AAA AAT TTA TTA AAT TAT ATA GAT GAA AAT AAA TTA TAT 2448 Thr Leu Lys Lys Asn Leu Leu Asn Tyr Ile Asp Glu Asn Lys Leu Tyr 805 810 815 TTA ATT GGA AGT GTA GAA GAT GAA AAA TCA AAA GTA GAT AAA TAC TTG 2496 Leu Ile Gly Ser Val Glu Asp Glu Lys Ser Lys Val Asp Lys Tyr Leu 820 825 830 AAA ACC ATT ATA CCA TTT GAT CTT TCA ACG TAT TCT AAT ATT GAA ATA 2544 Lys Thr Ile Ile Pro Phe Asp Leu Ser Thr Tyr Ser Asn Ile Glu Ile 835 840 845 CTA ATA AAA ATA TTT AAT AAA TAT AAT AGC GAA ATT TTA AAT AAT ATT 2592 Leu Ile Lys Ile Phe Asn Lys Tyr Asn Ser Glu Ile Leu Asn Asn Ile 850 855 860 ATC TTA AAT TTA AGA TAT AGA GAT AAT AAT TTA ATA GAT TTA TCA GGA 2640 Ile Leu Asn Leu Arg Tyr Arg Asp Asn Asn Leu Ile Asp Leu Ser Gly 865 870 875 880 TAT GGA GCA AAG GTA GAG GTA TAT GAT GGG GTC AAG CTT AAT GAT AAA 2688 Tyr Gly Ala Lys Val Glu Val Tyr Asp Gly Val Lys Leu Asn Asp Lys 885 890 895 AAT CAA TTT AAA TTA ACT AGT TCA GCA GAT AGT AAG ATT AGA GTC ACT 2736 Asn Gln Phe Lys Leu Thr Ser Ser Ala Asp Ser Lys Ile Arg Val Thr 900 905 910 CAA AAT CAG AAT ATT ATA TTT AAT AGT ATG TTC CTT GAT TTT AGC GTT 2784 Gln Asn Gln Asn Ile Ile Phe Asn Ser Met Phe Leu Asp Phe Ser Val 915 920 925 AGC TTT TGG ATA AGG ATA CCT AAA TAT AGG AAT GAT GAT ATA CAA AAT 2832 Ser Phe Trp Ile Arg Ile Pro Lys Tyr Arg Asn Asp Asp Ile Gln Asn 930 935 940 TAT ATT CAT AAT GAA TAT ACG ATA ATT AAT TGT ATG AAA AAT AAT TCA 2880 Tyr Ile His Asn Glu Tyr Thr Ile Ile Asn Cys Met Lys Asn Asn Ser 945 950 955 960 GGC TGG AAA ATA TCT ATT AGG GGT AAT AGG ATA ATA TGG ACC TTA ATT 2928 Gly Trp Lys Ile Ser Ile Arg Gly Asn Arg Ile Ile Trp Thr Leu Ile 965 970 975 GAT ATA AAT GGA AAA ACC AAA TCA GTA TTT TTT GAA TAT AAC ATA AGA 2976 Asp Ile Asn Gly Lys Thr Lys Ser Val Phe Phe Glu Tyr Asn Ile Arg 980 985 990 GAA GAT ATA TCA GAG TAT ATA AAT AGA TGG TTT TTT GTA ACT ATT ACT 3024 Glu Asp Ile Ser Glu Tyr Ile Asn Arg Trp Phe Phe Val Thr Ile Thr 995 1000 1005 AAT AAT TTG GAT AAT GCT AAA ATT TAT ATT AAT GGC ACG TTA GAA TCA 3072 Asn Asn Leu Asp Asn Ala Lys Ile Tyr Ile Asn Gly Thr Leu Glu Ser 1010 1015 1020 AAT ATG GAT ATT AAA GAT ATA GGA GAA GTT ATT GTT AAT GGT GAA ATA 3120 Asn Met Asp Ile Lys Asp Ile Gly Glu Val Ile Val Asn Gly Glu Ile 1025 1030 1035 1040 ACA TTT AAA TTA GAT GGT GAT GTA GAT AGA ACA CAA TTT ATT TGG ATG 3168 Thr Phe Lys Leu Asp Gly Asp Val Asp Arg Thr Gln Phe Ile Trp Met 1045 1050 1055 AAA TAT TTT AGT ATT TTT AAT ACG CAA TTA AAT CAA TCA AAT ATT AAA 3216 Lys Tyr Phe Ser Ile Phe Asn Thr Gln Leu Asn Gln Ser Asn Ile Lys 1060 1065 1070 GAG ATA TAT AAA ATT CAA TCA TAT AGC GAA TAC TTA AAA GAT TTT TGG 3264 Glu Ile Tyr Lys Ile Gln Ser Tyr Ser Glu Tyr Leu Lys Asp Phe Trp 1075 1080 1085 GGA AAT CCT TTA ATG TAT AAT AAA GAA TAT TAT ATG TTT AAT GCG GGG 3312 Gly Asn Pro Leu Met Tyr Asn Lys Glu Tyr Tyr Met Phe Asn Ala Gly 1090 1095 1100 AAT AAA AAT TCA TAT ATT AAA CTA GTG AAA GAT TCA TCT GTA GGT GAA 3360 Asn Lys Asn Ser Tyr Ile Lys Leu Val Lys Asp Ser Ser Val Gly Glu 1105 1110 1115 1120 ATA TTA ATA CGT AGC AAA TAT AAT CAG AAT TCC AAT TAT ATA AAT TAT 3408 Ile Leu Ile Arg Ser Lys Tyr Asn Gln Asn Ser Asn Tyr Ile Asn Tyr 1125 1130 1135 AGA AAT TTA TAT ATT GGA GAA AAA TTT ATT ATA AGA AGA GAG TCA AAT 3456 Arg Asn Leu Tyr Ile Gly Glu Lys Phe Ile Ile Arg Arg Glu Ser Asn 1140 1145 1150 TCT CAA TCT ATA AAT GAT GAT ATA GTT AGA AAA GAA GAT TAT ATA CAT 3504 Ser Gln Ser Ile Asn Asp Asp Ile Val Arg Lys Glu Asp Tyr Ile His 1155 1160 1165 CTA GAT TTG GTA CTT CAC CAT GAA GAG TGG AGA GTA TAT GCC TAT AAA 3552 Leu Asp Leu Val Leu His His Glu Glu Trp Arg Val Tyr Ala Tyr Lys 1170 1175 1180 TAT TTT AAG GAA CAG GAA GAA AAA TTG TTT TTA TCT ATT ATA AGT GAT 3600 Tyr Phe Lys Glu Gln Glu Glu Lys Leu Phe Leu Ser Ile Ile Ser Asp 1185 1190 1195 1200 TCT AAT GAA TTT TAT AAG ACT ATA GAA ATA AAA GAA TAT GAT GAA CAG 3648 Ser Asn Glu Phe Tyr Lys Thr Ile Glu Ile Lys Glu Tyr Asp Glu Gln 1205 1210 1215 CCA TCA TAT AGT TGT CAG TTG CTT TTT AAA AAA GAT GAA GAA AGT ACT 3696 Pro Ser Tyr Ser Cys Gln Leu Leu Phe Lys Lys Asp Glu Glu Ser Thr 1220 1225 1230 GAT GAT ATA GGA TTG ATT GGT ATT CAT CGT TTC TAC GAA TCT GGA GTT 3744 Asp Asp Ile Gly Leu Ile Gly Ile His Arg Phe Tyr Glu Ser Gly Val 1235 1240 1245 TTA CGT AAA AAG TAT AAA GAT TAT TTT TGT ATA AGT AAA TGG TAC TTA 3792 Leu Arg Lys Lys Tyr Lys Asp Tyr Phe Cys Ile Ser Lys Trp Tyr Leu 1250 1255 1260 AAA GAG GTA AAA AGG AAA CCA TAT AAG TCA AAT TTG GGA TGT AAT TGG 3840 Lys Glu Val Lys Arg Lys Pro Tyr Lys Ser Asn Leu Gly Cys Asn Trp 1265 1270 1275 1280 CAG TTT ATT CCT AAA GAT GAA GGG TGG ACT GAA TAA 3876 Gln Phe Ile Pro Lys Asp Glu Gly Trp Thr Glu 1285 1290

40 Met Pro Val Thr Ile Asn Asn Phe Asn Tyr Asn Asp Pro Ile Asp Asn 1 5 10 15 Asp Asn Ile Ile Met Met Glu Pro Pro Phe Ala Arg Gly Thr Gly Arg 20 25 30 Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Ile Pro Glu 35 40 45 Arg Tyr Thr Phe Gly Tyr Lys Pro Glu Asp Phe Asn Lys Ser Ser Gly 50 55 60 Ile Phe Asn Arg Asp Val Cys Glu Tyr Tyr Asp Pro Asp Tyr Leu Asn 65 70 75 80 Thr Asn Asp Lys Lys Asn Ile Phe Phe Gln Thr Leu Ile Lys Leu Phe 85 90 95 Asn Arg Ile Lys Ser Lys Pro Leu Gly Glu Lys Leu Leu Glu Met Ile 100 105 110 Ile Asn Gly Ile Pro Tyr Leu Gly Asp Arg Arg Val Pro Leu Glu Glu 115 120 125 Phe Asn Thr Asn Ile Ala Ser Val Thr Val Asn Lys Leu Ile Ser Asn 130 135 140 Pro Gly Glu Val Glu Arg Lys Lys Gly Ile Phe Ala Asn Leu Ile Ile 145 150 155 160 Phe Gly Pro Gly Pro Val Leu Asn Glu Asn Glu Thr Ile Asp Ile Gly 165 170 175 Ile Gln Asn His Phe Ala Ser Arg Glu Gly Phe Gly Gly Ile Met Gln 180 185 190 Met Lys Phe Cys Pro Glu Tyr Val Ser Val Phe Asn Asn Val Gln Glu 195 200 205 Asn Lys Gly Ala Ser Ile Phe Asn Arg Arg Gly Tyr Phe Ser Asp Pro 210 215 220 Ala Leu Ile Leu Met His Glu Leu Ile His Val Leu His Gly Leu Tyr 225 230 235 240 Gly Ile Lys Val Asp Asp Leu Pro Ile Val Pro Asn Glu Lys Lys Phe 245 250 255 Phe Met Gln Ser Thr Asp Thr Ile Gln Ala Glu Glu Leu Tyr Thr Phe 260 265 270 Gly Gly Gln Asp Pro Ser Ile Ile Ser Pro Ser Thr Asp Lys Ser Ile 275 280 285 Tyr Asp Lys Val Leu Gln Asn Phe Arg Gly Ile Val Asp Arg Leu Asn 290 295 300 Lys Val Leu Val Cys Ile Ser Asp Pro Asn Ile Asn Ile Asn Ile Tyr 305 310 315 320 Lys Asn Lys Phe Lys Asp Lys Tyr Lys Phe Val Glu Asp Ser Glu Gly 325 330 335 Lys Tyr Ser Ile Asp Val Glu Ser Phe Asn Lys Leu Tyr Lys Ser Leu 340 345 350 Met Leu Gly Phe Thr Glu Ile Asn Ile Ala Glu Asn Tyr Lys Ile Lys 355 360 365 Thr Arg Ala Ser Tyr Phe Ser Asp Ser Leu Pro Pro Val Lys Ile Lys 370 375 380 Asn Leu Leu Asp Asn Glu Ile Tyr Thr Ile Glu Glu Gly Phe Asn Ile 385 390 395 400 Ser Asp Lys Asn Met Gly Lys Glu Tyr Arg Gly Gln Asn Lys Ala Ile 405 410 415 Asn Lys Gln Ala Tyr Glu Glu Ile Ser Lys Glu His Leu Ala Val Tyr 420 425 430 Lys Ile Gln Met Cys Lys Ser Val Lys Val Pro Gly Ile Cys Ile Asp 435 440 445 Val Asp Asn Glu Asn Leu Phe Phe Ile Ala Asp Lys Asn Ser Phe Ser 450 455 460 Asp Asp Leu Ser Lys Asn Glu Arg Val Glu Tyr Asn Thr Gln Asn Asn 465 470 475 480 Tyr Ile Gly Asn Asp Phe Pro Ile Asn Glu Leu Ile Leu Asp Thr Asp 485 490 495 Leu Ile Ser Lys Ile Glu Leu Pro Ser Glu Asn Thr Glu Ser Leu Thr 500 505 510 Asp Phe Asn Val Asp Val Pro Val Tyr Glu Lys Gln Pro Ala Ile Lys 515 520 525 Lys Val Phe Thr Asp Glu Asn Thr Ile Phe Gln Tyr Leu Tyr Ser Gln 530 535 540 Thr Phe Pro Leu Asn Ile Arg Asp Ile Ser Leu Thr Ser Ser Phe Asp 545 550 555 560 Asp Ala Leu Leu Val Ser Ser Lys Val Tyr Ser Phe Phe Ser Met Asp 565 570 575 Tyr Ile Lys Thr Ala Asn Lys Val Val Glu Ala Gly Leu Phe Ala Gly 580 585 590 Trp Val Lys Gln Ile Val Asp Asp Phe Val Ile Glu Ala Asn Lys Ser 595 600 605 Ser Thr Met Asp Lys Ile Ala Asp Ile Ser Leu Ile Val Pro Tyr Ile 610 615 620 Gly Leu Ala Leu Asn Val Gly Asp Glu Thr Ala Lys Gly Asn Phe Glu 625 630 635 640 Ser Ala Phe Glu Ile Ala Gly Ser Ser Ile Leu Leu Glu Phe Ile Pro 645 650 655 Glu Leu Leu Ile Pro Val Val Gly Val Phe Leu Leu Glu Ser Tyr Ile 660 665 670 Asp Asn Lys Asn Lys Ile Ile Lys Thr Ile Asp Asn Ala Leu Thr Lys 675 680 685 Arg Val Glu Lys Trp Ile Asp Met Tyr Gly Leu Ile Val Ala Gln Trp 690 695 700 Leu Ser Thr Val Asn Thr Gln Phe Tyr Thr Ile Lys Glu Gly Met Tyr 705 710 715 720 Lys Ala Leu Asn Tyr Gln Ala Gln Ala Leu Glu Glu Ile Ile Lys Tyr 725 730 735 Lys Tyr Asn Ile Tyr Ser Glu Glu Glu Lys Ser Asn Ile Asn Ile Asn 740 745 750 Phe Asn Asp Ile Asn Ser Lys Leu Asn Asp Gly Ile Asn Gln Ala Met 755 760 765 Asp Asn Ile Asn Asp Phe Ile Asn Glu Cys Ser Val Ser Tyr Leu Met 770 775 780 Lys Lys Met Ile Pro Leu Ala Val Lys Lys Leu Leu Asp Phe Asp Asn 785 790 795 800 Thr Leu Lys Lys Asn Leu Leu Asn Tyr Ile Asp Glu Asn Lys Leu Tyr 805 810 815 Leu Ile Gly Ser Val Glu Asp Glu Lys Ser Lys Val Asp Lys Tyr Leu 820 825 830 Lys Thr Ile Ile Pro Phe Asp Leu Ser Thr Tyr Ser Asn Ile Glu Ile 835 840 845 Leu Ile Lys Ile Phe Asn Lys Tyr Asn Ser Glu Ile Leu Asn Asn Ile 850 855 860 Ile Leu Asn Leu Arg Tyr Arg Asp Asn Asn Leu Ile Asp Leu Ser Gly 865 870 875 880 Tyr Gly Ala Lys Val Glu Val Tyr Asp Gly Val Lys Leu Asn Asp Lys 885 890 895 Asn Gln Phe Lys Leu Thr Ser Ser Ala Asp Ser Lys Ile Arg Val Thr 900 905 910 Gln Asn Gln Asn Ile Ile Phe Asn Ser Met Phe Leu Asp Phe Ser Val 915 920 925 Ser Phe Trp Ile Arg Ile Pro Lys Tyr Arg Asn Asp Asp Ile Gln Asn 930 935 940 Tyr Ile His Asn Glu Tyr Thr Ile Ile Asn Cys Met Lys Asn Asn Ser 945 950 955 960 Gly Trp Lys Ile Ser Ile Arg Gly Asn Arg Ile Ile Trp Thr Leu Ile 965 970 975 Asp Ile Asn Gly Lys Thr Lys Ser Val Phe Phe Glu Tyr Asn Ile Arg 980 985 990 Glu Asp Ile Ser Glu Tyr Ile Asn Arg Trp Phe Phe Val Thr Ile Thr 995 1000 1005 Asn Asn Leu Asp Asn Ala Lys Ile Tyr Ile Asn Gly Thr Leu Glu Ser 1010 1015 1020 Asn Met Asp Ile Lys Asp Ile Gly Glu Val Ile Val Asn Gly Glu Ile 1025 1030 1035 1040 Thr Phe Lys Leu Asp Gly Asp Val Asp Arg Thr Gln Phe Ile Trp Met 1045 1050 1055 Lys Tyr Phe Ser Ile Phe Asn Thr Gln Leu Asn Gln Ser Asn Ile Lys 1060 1065 1070 Glu Ile Tyr Lys Ile Gln Ser Tyr Ser Glu Tyr Leu Lys Asp Phe Trp 1075 1080 1085 Gly Asn Pro Leu Met Tyr Asn Lys Glu Tyr Tyr Met Phe Asn Ala Gly 1090 1095 1100 Asn Lys Asn Ser Tyr Ile Lys Leu Val Lys Asp Ser Ser Val Gly Glu 1105 1110 1115 1120 Ile Leu Ile Arg Ser Lys Tyr Asn Gln Asn Ser Asn Tyr Ile Asn Tyr 1125 1130 1135 Arg Asn Leu Tyr Ile Gly Glu Lys Phe Ile Ile Arg Arg Glu Ser Asn 1140 1145 1150 Ser Gln Ser Ile Asn Asp Asp Ile Val Arg Lys Glu Asp Tyr Ile His 1155 1160 1165 Leu Asp Leu Val Leu His His Glu Glu Trp Arg Val Tyr Ala Tyr Lys 1170 1175 1180 Tyr Phe Lys Glu Gln Glu Glu Lys Leu Phe Leu Ser Ile Ile Ser Asp 1185 1190 1195 1200 Ser Asn Glu Phe Tyr Lys Thr Ile Glu Ile Lys Glu Tyr Asp Glu Gln 1205 1210 1215 Pro Ser Tyr Ser Cys Gln Leu Leu Phe Lys Lys Asp Glu Glu Ser Thr 1220 1225 1230 Asp Asp Ile Gly Leu Ile Gly Ile His Arg Phe Tyr Glu Ser Gly Val 1235 1240 1245 Leu Arg Lys Lys Tyr Lys Asp Tyr Phe Cys Ile Ser Lys Trp Tyr Leu 1250 1255 1260 Lys Glu Val Lys Arg Lys Pro Tyr Lys Ser Asn Leu Gly Cys Asn Trp 1265 1270 1275 1280 Gln Phe Ile Pro Lys Asp Glu Gly Trp Thr Glu 1285 1290

41 ATG CCA GTT ACA ATA AAT AAT TTT AAT TAT AAT GAT CCT ATT GAT AAT 48 Met Pro Val Thr Ile Asn Asn Phe Asn Tyr Asn Asp Pro Ile Asp Asn 1 5 10 15 AAT AAT ATT ATT ATG ATG GAG CCT CCA TTT GCG AGA GGT ACG GGG AGA 96 Asn Asn Ile Ile Met Met Glu Pro Pro Phe Ala Arg Gly Thr Gly Arg 20 25 30 TAT TAT AAA GCT TTT AAA ATC ACA GAT CGT ATT TGG ATA ATA CCG GAA 144 Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Ile Pro Glu 35 40 45 AGA TAT ACT TTT GGA TAT AAA CCT GAG GAT TTT AAT AAA AGT TCC GGT 192 Arg Tyr Thr Phe Gly Tyr Lys Pro Glu Asp Phe Asn Lys Ser Ser Gly 50 55 60 ATT TTT AAT AGA GAT GTT TGT GAA TAT TAT GAT CCA GAT TAC TTA AAT 240 Ile Phe Asn Arg Asp Val Cys Glu Tyr Tyr Asp Pro Asp Tyr Leu Asn 65 70 75 80 ACT AAT GAT AAA AAG AAT ATA TTT TTA CAA ACA ATG ATC AAG TTA TTT 288 Thr Asn Asp Lys Lys Asn Ile Phe Leu Gln Thr Met Ile Lys Leu Phe 85 90 95 AAT AGA ATC AAA TCA AAA CCA TTG GGT GAA AAG TTA TTA GAG ATG ATT 336 Asn Arg Ile Lys Ser Lys Pro Leu Gly Glu Lys Leu Leu Glu Met Ile 100 105 110 ATA AAT GGT ATA CCT TAT CTT GGA GAT AGA CGT GTT CCA CTC GAA GAG 384 Ile Asn Gly Ile Pro Tyr Leu Gly Asp Arg Arg Val Pro Leu Glu Glu 115 120 125 TTT AAC ACA AAC ATT GCT AGT GTA ACT GTT AAT AAA TTA ATC AGT AAT 432 Phe Asn Thr Asn Ile Ala Ser Val Thr Val Asn Lys Leu Ile Ser Asn 130 135 140 CCA GGA GAA GTG GAG CGA AAA AAA GGT ATT TTC GCA AAT TTA ATA ATA 480 Pro Gly Glu Val Glu Arg Lys Lys Gly Ile Phe Ala Asn Leu Ile Ile 145 150 155 160 TTT GGA CCT GGG CCA GTT TTA AAT GAA AAT GAG ACT ATA GAT ATA GGT 528 Phe Gly Pro Gly Pro Val Leu Asn Glu Asn Glu Thr Ile Asp Ile Gly 165 170 175 ATA CAA AAT CAT TTT GCA TCA AGG GAA GGC TTC GGG GGT ATA ATG CAA 576 Ile Gln Asn His Phe Ala Ser Arg Glu Gly Phe Gly Gly Ile Met Gln 180 185 190 ATG AAG TTT TGC CCA GAA TAT GTA AGC GTA TTT AAT AAT GTT CAA GAA 624 Met Lys Phe Cys Pro Glu Tyr Val Ser Val Phe Asn Asn Val Gln Glu 195 200 205 AAC AAA GGC GCA AGT ATA TTT AAT AGA CGT GGA TAT TTT TCA GAT CCA 672 Asn Lys Gly Ala Ser Ile Phe Asn Arg Arg Gly Tyr Phe Ser Asp Pro 210 215 220 GCC TTG ATA TTA ATG CAT GAA CTT ATA CAT GTT TTA CAT GGA TTA TAT 720 Ala Leu Ile Leu Met His Glu Leu Ile His Val Leu His Gly Leu Tyr 225 230 235 240 GGC ATT AAA GTA GAT GAT TTA CCA ATT GTA CCA AAT GAA AAA AAA TTT 768 Gly Ile Lys Val Asp Asp Leu Pro Ile Val Pro Asn Glu Lys Lys Phe 245 250 255 TTT ATG CAA TCT ACA GAT GCT ATA CAG GCA GAA GAA CTA TAT ACA TTT 816 Phe Met Gln Ser Thr Asp Ala Ile Gln Ala Glu Glu Leu Tyr Thr Phe 260 265 270 GGA GGA CAA GAT CCC AGC ATC ATA ACT CCT TCT ACG GAT AAA AGT ATC 864 Gly Gly Gln Asp Pro Ser Ile Ile Thr Pro Ser Thr Asp Lys Ser Ile 275 280 285 TAT GAT AAA GTT TTG CAA AAT TTT AGA GGG ATA GTT GAT AGA CTT AAC 912 Tyr Asp Lys Val Leu Gln Asn Phe Arg Gly Ile Val Asp Arg Leu Asn 290 295 300 AAG GTT TTA GTT TGC ATA TCA GAT CCT AAC ATT AAT ATT AAT ATA TAT 960 Lys Val Leu Val Cys Ile Ser Asp Pro Asn Ile Asn Ile Asn Ile Tyr 305 310 315 320 AAA AAT AAA TTT AAA GAT AAA TAT AAA TTC GTT GAA GAT TCT GAG GGA 1008 Lys Asn Lys Phe Lys Asp Lys Tyr Lys Phe Val Glu Asp Ser Glu Gly 325 330 335 AAA TAT AGT ATA GAT GTA GAA AGT TTT GAT AAA TTA TAT AAA AGC TTA 1056 Lys Tyr Ser Ile Asp Val Glu Ser Phe Asp Lys Leu Tyr Lys Ser Leu 340 345 350 ATG TTT GGT TTT ACA GAA ACT AAT ATA GCA GAA AAT TAT AAA ATA AAA 1104 Met Phe Gly Phe Thr Glu Thr Asn Ile Ala Glu Asn Tyr Lys Ile Lys 355 360 365 ACT AGA GCT TCT TAT TTT AGT GAT TCC TTA CCA CCA GTA AAA ATA AAA 1152 Thr Arg Ala Ser Tyr Phe Ser Asp Ser Leu Pro Pro Val Lys Ile Lys 370 375 380 AAT TTA TTA GAT AAT GAA ATC TAT ACT ATA GAG GAA GGG TTT AAT ATA 1200 Asn Leu Leu Asp Asn Glu Ile Tyr Thr Ile Glu Glu Gly Phe Asn Ile 385 390 395 400 TCT GAT AAA GAT ATG GAA AAA GAA TAT AGA GGT CAG AAT AAA GCT ATA 1248 Ser Asp Lys Asp Met Glu Lys Glu Tyr Arg Gly Gln Asn Lys Ala Ile 405 410 415 AAT AAA CAA GCT TAT GAA GAA ATT AGC AAG GAG CAT TTG GCT GTA TAT 1296 Asn Lys Gln Ala Tyr Glu Glu Ile Ser Lys Glu His Leu Ala Val Tyr 420 425 430 AAG ATA CAA ATG TGT AAA AGT GTT AAA GCT CCA GGA ATA TGT ATT GAT 1344 Lys Ile Gln Met Cys Lys Ser Val Lys Ala Pro Gly Ile Cys Ile Asp 435 440 445 GTT GAT AAT GAA GAT TTG TTC TTT ATA GCT GAT AAA AAT AGT TTT TCA 1392 Val Asp Asn Glu Asp Leu Phe Phe Ile Ala Asp Lys Asn Ser Phe Ser 450 455 460 GAT GAT TTA TCT AAA AAC GAA AGA ATA GAA TAT AAT ACA CAG AGT AAT 1440 Asp Asp Leu Ser Lys Asn Glu Arg Ile Glu Tyr Asn Thr Gln Ser Asn 465 470 475 480 TAT ATA GAA AAT GAC TTC CCT ATA AAT GAA TTA ATT TTA GAT ACT GAT 1488 Tyr Ile Glu Asn Asp Phe Pro Ile Asn Glu Leu Ile Leu Asp Thr Asp 485 490 495 TTA ATA AGT AAA ATA GAA TTA CCA AGT GAA AAT ACA GAA TCA CTT ACT 1536 Leu Ile Ser Lys Ile Glu Leu Pro Ser Glu Asn Thr Glu Ser Leu Thr 500 505 510 GAT TTT AAT GTA GAT GTT CCA GTA TAT GAA AAA CAA CCC GCT ATA AAA 1584 Asp Phe Asn Val Asp Val Pro Val Tyr Glu Lys Gln Pro Ala Ile Lys 515 520 525 AAA ATT TTT ACA GAT GAA AAT ACC ATC TTT CAA TAT TTA TAC TCT CAG 1632 Lys Ile Phe Thr Asp Glu Asn Thr Ile Phe Gln Tyr Leu Tyr Ser Gln 530 535 540 ACA TTT CTC TTA GAT ATA AGA GAT ATA AGT TTA ACA TCT TCA TTT GAT 1680 Thr Phe Leu Leu Asp Ile Arg Asp Ile Ser Leu Thr Ser Ser Phe Asp 545 550 555 560 GAT GCA TTA TTA TTT TCT AAC AAA GTT TAT TCA TTT TTT TCT ATG GAT 1728 Asp Ala Leu Leu Phe Ser Asn Lys Val Tyr Ser Phe Phe Ser Met Asp 565 570 575 TAT ATT AAA ACT GCT AAT AAA GTG GTA GAA GCA GGA TTA TTT GCA GGT 1776 Tyr Ile Lys Thr Ala Asn Lys Val Val Glu Ala Gly Leu Phe Ala Gly 580 585 590 TGG GTG AAA CAG ATA GTA AAT GAT TTT GTA ATC GAA GCT AAT AAA AGC 1824 Trp Val Lys Gln Ile Val Asn Asp Phe Val Ile Glu Ala Asn Lys Ser 595 600 605 AAT ACT ATG GAT AAA ATT GCA GAT ATA TCT CTA ATT GTT CCT TAT ATA 1872 Asn Thr Met Asp Lys Ile Ala Asp Ile Ser Leu Ile Val Pro Tyr Ile 610 615 620 GGA TTA GCT TTA AAT GTA GGA AAT GAA ACA GCT AAA GGA AAT TTT GAA 1920 Gly Leu Ala Leu Asn Val Gly Asn Glu Thr Ala Lys Gly Asn Phe Glu 625 630 635 640 AAT GCT TTT GAG ATT GCA GGA GCC AGT ATT CTA CTA GAA TTT ATA CCA 1968 Asn Ala Phe Glu Ile Ala Gly Ala Ser Ile Leu Leu Glu Phe Ile Pro 645 650 655 GAA CTT TTA ATA CCT GTA GTT GGA GCC TTT TTA TTA GAA TCA TAT ATT 2016 Glu Leu Leu Ile Pro Val Val Gly Ala Phe Leu Leu Glu Ser Tyr Ile 660 665 670 GAC AAT AAA AAT AAA ATT ATT AAA ACA ATA GAT AAT GCT TTA ACT AAA 2064 Asp Asn Lys Asn Lys Ile Ile Lys Thr Ile Asp Asn Ala Leu Thr Lys 675 680 685 AGA AAT GAA AAA TGG AGT GAT ATG TAC GGA TTA ATA GTA GCG CAA TGG 2112 Arg Asn Glu Lys Trp Ser Asp Met Tyr Gly Leu Ile Val Ala Gln Trp 690 695 700 CTC TCA ACA GTT AAT ACT CAA TTT TAT ACA ATA AAA GAG GGA ATG TAT 2160 Leu Ser Thr Val Asn Thr Gln Phe Tyr Thr Ile Lys Glu Gly Met Tyr 705 710 715 720 AAG GCT TTA AAT TAT CAA GCA CAA GCA TTG GAA GAA ATA ATA AAA TAC 2208 Lys Ala Leu Asn Tyr Gln Ala Gln Ala Leu Glu Glu Ile Ile Lys Tyr 725 730 735 AGA TAT AAT ATA TAT TCT GAA AAA GAA AAG TCA AAT ATT AAC ATC GAT 2256 Arg Tyr Asn Ile Tyr Ser Glu Lys Glu Lys Ser Asn Ile Asn Ile Asp 740 745 750 TTT AAT GAT ATA AAT TCT AAA CTT AAT GAG GGT ATT AAC CAA GCT ATA 2304 Phe Asn Asp Ile Asn Ser Lys Leu Asn Glu Gly Ile Asn Gln Ala Ile 755 760 765 GAT AAT ATA AAT AAT TTT ATA AAT GGA TGT TCT GTA TCA TAT TTA ATG 2352 Asp Asn Ile Asn Asn Phe Ile Asn Gly Cys Ser Val Ser Tyr Leu Met 770 775 780 AAA AAA ATG ATT CCA TTA GCT GTA GAA AAA TTA CTA GAC TTT GAT AAT 2400 Lys Lys Met Ile Pro Leu Ala Val Glu Lys Leu Leu Asp Phe Asp Asn 785 790 795 800 ACT CTC AAA AAA AAT TTG TTA AAT TAT ATA GAT GAA AAT AAA TTA TAT 2448 Thr Leu Lys Lys Asn Leu Leu Asn Tyr Ile Asp Glu Asn Lys Leu Tyr 805 810 815 TTG ATT GGA AGT GCA GAA TAT GAA AAA TCA AAA GTA AAT AAA TAC TTG 2496 Leu Ile Gly Ser Ala Glu Tyr Glu Lys Ser Lys Val Asn Lys Tyr Leu 820 825 830 AAA ACC ATT ATG CCG TTT GAT CTT TCA ATA TAT ACC AAT GAT ACA ATA 2544 Lys Thr Ile Met Pro Phe Asp Leu Ser Ile Tyr Thr Asn Asp Thr Ile 835 840 845 CTA ATA GAA ATG TTT AAT AAA TAT AAT AGC GAA ATT TTA AAT AAT ATT 2592 Leu Ile Glu Met Phe Asn Lys Tyr Asn Ser Glu Ile Leu Asn Asn Ile 850 855 860 ATC TTA AAT TTA AGA TAT AAG GAT AAT AAT TTA ATA GAT TTA TCA GGA 2640 Ile Leu Asn Leu Arg Tyr Lys Asp Asn Asn Leu Ile Asp Leu Ser Gly 865 870 875 880 TAT GGG GCA AAG GTA GAG GTA TAT GAT GGA GTC GAG CTT AAT GAT AAA 2688 Tyr Gly Ala Lys Val Glu Val Tyr Asp Gly Val Glu Leu Asn Asp Lys 885 890 895 AAT CAA TTT AAA TTA ACT AGT TCA GCA AAT AGT AAG ATT AGA GTG ACT 2736 Asn Gln Phe Lys Leu Thr Ser Ser Ala Asn Ser Lys Ile Arg Val Thr 900 905 910 CAA AAT CAG AAT ATC ATA TTT AAT AGT GTG TTC CTT GAT TTT AGC GTT 2784 Gln Asn Gln Asn Ile Ile Phe Asn Ser Val Phe Leu Asp Phe Ser Val 915 920 925 AGC TTT TGG ATA AGA ATA CCT AAA TAT AAG AAT GAT GGT ATA CAA AAT 2832 Ser Phe Trp Ile Arg Ile Pro Lys Tyr Lys Asn Asp Gly Ile Gln Asn 930 935 940 TAT ATT CAT AAT GAA TAT ACA ATA ATT AAT TGT ATG AAA AAT AAT TCG 2880 Tyr Ile His Asn Glu Tyr Thr Ile Ile Asn Cys Met Lys Asn Asn Ser 945 950 955 960 GGC TGG AAA ATA TCT ATT AGG GGT AAT AGG ATA ATA TGG ACT TTA ATT 2928 Gly Trp Lys Ile Ser Ile Arg Gly Asn Arg Ile Ile Trp Thr Leu Ile 965 970 975 GAT ATA AAT GGA AAA ACC AAA TCG GTA TTT TTT GAA TAT AAC ATA AGA 2976 Asp Ile Asn Gly Lys Thr Lys Ser Val Phe Phe Glu Tyr Asn Ile Arg 980 985 990 GAA GAT ATA TCA GAG TAT ATA AAT AGA TGG TTT TTT GTA ACT ATT ACT 3024 Glu Asp Ile Ser Glu Tyr Ile Asn Arg Trp Phe Phe Val Thr Ile Thr 995 1000 1005 AAT AAT TTG AAT AAC GCT AAA ATT TAT ATT AAT GGT AAG CTA GAA TCA 3072 Asn Asn Leu Asn Asn Ala Lys Ile Tyr Ile Asn Gly Lys Leu Glu Ser 1010 1015 1020 AAT ACA GAT ATT AAA GAT ATA AGA GAA GTT ATT GCT AAT GGT GAA ATA 3120 Asn Thr Asp Ile Lys Asp Ile Arg Glu Val Ile Ala Asn Gly Glu Ile 1025 1030 1035 1040 ATA TTT AAA TTA GAT GGT GAT ATA GAT AGA ACA CAA TTT ATT TGG ATG 3168 Ile Phe Lys Leu Asp Gly Asp Ile Asp Arg Thr Gln Phe Ile Trp Met 1045 1050 1055 AAA TAT TTC AGT ATT TTT AAT ACG GAA TTA AGT CAA TCA AAT ATT GAA 3216 Lys Tyr Phe Ser Ile Phe Asn Thr Glu Leu Ser Gln Ser Asn Ile Glu 1060 1065 1070 GAA AGA TAT AAA ATT CAA TCA TAT AGC GAA TAT TTA AAA GAT TTT TGG 3264 Glu Arg Tyr Lys Ile Gln Ser Tyr Ser Glu Tyr Leu Lys Asp Phe Trp 1075 1080 1085 GGA AAT CCT TTA ATG TAC AAT AAA GAA TAT TAT ATG TTT AAT GCG GGG 3312 Gly Asn Pro Leu Met Tyr Asn Lys Glu Tyr Tyr Met Phe Asn Ala Gly 1090 1095 1100 AAT AAA AAT TCA TAT ATT AAA CTA AAG AAA GAT TCA CCT GTA GGT GAA 3360 Asn Lys Asn Ser Tyr Ile Lys Leu Lys Lys Asp Ser Pro Val Gly Glu 1105 1110 1115 1120 ATT TTA ACA CGT AGC AAA TAT AAT CAA AAT TCT AAA TAT ATA AAT TAT 3408 Ile Leu Thr Arg Ser Lys Tyr Asn Gln Asn Ser Lys Tyr Ile Asn Tyr 1125 1130 1135 AGA GAT TTA TAT ATT GGA GAA AAA TTT ATT ATA AGA AGA AAG TCA AAT 3456 Arg Asp Leu Tyr Ile Gly Glu Lys Phe Ile Ile Arg Arg Lys Ser Asn 1140 1145 1150 TCT CAA TCT ATA AAT GAT GAT ATA GTT AGA AAA GAA GAT TAT ATA TAT 3504 Ser Gln Ser Ile Asn Asp Asp Ile Val Arg Lys Glu Asp Tyr Ile Tyr 1155 1160 1165 CTA GAT TTT TTT AAT TTA AAT CAA GAG TGG AGA GTA TAT ACC TAT AAA 3552 Leu Asp Phe Phe Asn Leu Asn Gln Glu Trp Arg Val Tyr Thr Tyr Lys 1170 1175 1180 TAT TTT AAG AAA GAG GAA GAA AAA TTG TTT TTA GCT CCT ATA AGT GAT 3600 Tyr Phe Lys Lys Glu Glu Glu Lys Leu Phe Leu Ala Pro Ile Ser Asp 1185 1190 1195 1200 TCT GAT GAG TTT TAC AAT ACT ATA CAA ATA AAA GAA TAT GAT GAA CAG 3648 Ser Asp Glu Phe Tyr Asn Thr Ile Gln Ile Lys Glu Tyr Asp Glu Gln 1205 1210 1215 CCA ACA TAT AGT TGT CAG TTG CTT TTT AAA AAA GAT GAA GAA AGT ACT 3696 Pro Thr Tyr Ser Cys Gln Leu Leu Phe Lys Lys Asp Glu Glu Ser Thr 1220 1225 1230 GAT GAG ATA GGA TTG ATT GGT ATT CAT CGT TTC TAC GAA TCT GGA ATT 3744 Asp Glu Ile Gly Leu Ile Gly Ile His Arg Phe Tyr Glu Ser Gly Ile 1235 1240 1245 GTA TTT GAA GAG TAT AAA GAT TAT TTT TGT ATA AGT AAA TGG TAC TTA 3792 Val Phe Glu Glu Tyr Lys Asp Tyr Phe Cys Ile Ser Lys Trp Tyr Leu 1250 1255 1260 AAA GAG GTA AAA AGG AAA CCA TAT AAT TTA AAA TTG GGA TGT AAT TGG 3840 Lys Glu Val Lys Arg Lys Pro Tyr Asn Leu Lys Leu Gly Cys Asn Trp 1265 1270 1275 1280 CAG TTT ATT CCT AAA GAT GAA GGG TGG ACT GAA TAA 3876 Gln Phe Ile Pro Lys Asp Glu Gly Trp Thr Glu 1285 1290

42 Met Pro Val Thr Ile Asn Asn Phe Asn Tyr Asn Asp Pro Ile Asp Asn 1 5 10 15 Asn Asn Ile Ile Met Met Glu Pro Pro Phe Ala Arg Gly Thr Gly Arg 20 25 30 Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Ile Pro Glu 35 40 45 Arg Tyr Thr Phe Gly Tyr Lys Pro Glu Asp Phe Asn Lys Ser Ser Gly 50 55 60 Ile Phe Asn Arg Asp Val Cys Glu Tyr Tyr Asp Pro Asp Tyr Leu Asn 65 70 75 80 Thr Asn Asp Lys Lys Asn Ile Phe Leu Gln Thr Met Ile Lys Leu Phe 85 90 95 Asn Arg Ile Lys Ser Lys Pro Leu Gly Glu Lys Leu Leu Glu Met Ile 100 105 110 Ile Asn Gly Ile Pro Tyr Leu Gly Asp Arg Arg Val Pro Leu Glu Glu 115 120 125 Phe Asn Thr Asn Ile Ala Ser Val Thr Val Asn Lys Leu Ile Ser Asn 130 135 140 Pro Gly Glu Val Glu Arg Lys Lys Gly Ile Phe Ala Asn Leu Ile Ile 145 150 155 160 Phe Gly Pro Gly Pro Val Leu Asn Glu Asn Glu Thr Ile Asp Ile Gly 165 170 175 Ile Gln Asn His Phe Ala Ser Arg Glu Gly Phe Gly Gly Ile Met Gln 180 185 190 Met Lys Phe Cys Pro Glu Tyr Val Ser Val Phe Asn Asn Val Gln Glu 195 200 205 Asn Lys Gly Ala Ser Ile Phe Asn Arg Arg Gly Tyr Phe Ser Asp Pro 210 215 220 Ala Leu Ile Leu Met His Glu Leu Ile His Val Leu His Gly Leu Tyr 225 230 235 240 Gly Ile Lys Val Asp Asp Leu Pro Ile Val Pro Asn Glu Lys Lys Phe 245 250 255 Phe Met Gln Ser Thr Asp Ala Ile Gln Ala Glu Glu Leu Tyr Thr Phe 260 265 270 Gly Gly Gln Asp Pro Ser Ile Ile Thr Pro Ser Thr Asp Lys Ser Ile 275 280 285 Tyr Asp Lys Val Leu Gln Asn Phe Arg Gly Ile Val Asp Arg Leu Asn 290 295 300 Lys Val Leu Val Cys Ile Ser Asp Pro Asn Ile Asn Ile Asn Ile Tyr 305 310 315 320 Lys Asn Lys Phe Lys Asp Lys Tyr Lys Phe Val Glu Asp Ser Glu Gly 325 330 335 Lys Tyr Ser Ile Asp Val Glu Ser Phe Asp Lys Leu Tyr Lys Ser Leu 340 345 350 Met Phe Gly Phe Thr Glu Thr Asn Ile Ala Glu Asn Tyr Lys Ile Lys 355 360 365 Thr Arg Ala Ser Tyr Phe Ser Asp Ser Leu Pro Pro Val Lys Ile Lys 370 375 380 Asn Leu Leu Asp Asn Glu Ile Tyr Thr Ile Glu Glu Gly Phe Asn Ile 385 390 395 400 Ser Asp Lys Asp Met Glu Lys Glu Tyr Arg Gly Gln Asn Lys Ala Ile 405 410 415 Asn Lys Gln Ala Tyr Glu Glu Ile Ser Lys Glu His Leu Ala Val Tyr 420 425 430 Lys Ile Gln Met Cys Lys Ser Val Lys Ala Pro Gly Ile Cys Ile Asp 435 440 445 Val Asp Asn Glu Asp Leu Phe Phe Ile Ala Asp Lys Asn Ser Phe Ser 450 455 460 Asp Asp Leu Ser Lys Asn Glu Arg Ile Glu Tyr Asn Thr Gln Ser Asn 465 470 475 480 Tyr Ile Glu Asn Asp Phe Pro Ile Asn Glu Leu Ile Leu Asp Thr Asp 485 490 495 Leu Ile Ser Lys Ile Glu Leu Pro Ser Glu Asn Thr Glu Ser Leu Thr 500 505 510 Asp Phe Asn Val Asp Val Pro Val Tyr Glu Lys Gln Pro Ala Ile Lys 515 520 525 Lys Ile Phe Thr Asp Glu Asn Thr Ile Phe Gln Tyr Leu Tyr Ser Gln 530 535 540 Thr Phe Leu Leu Asp Ile Arg Asp Ile Ser Leu Thr Ser Ser Phe Asp 545 550 555 560 Asp Ala Leu Leu Phe Ser Asn Lys Val Tyr Ser Phe Phe Ser Met Asp 565 570 575 Tyr Ile Lys Thr Ala Asn Lys Val Val Glu Ala Gly Leu Phe Ala Gly 580 585 590 Trp Val Lys Gln Ile Val Asn Asp Phe Val Ile Glu Ala Asn Lys Ser 595 600 605 Asn Thr Met Asp Lys Ile Ala Asp Ile Ser Leu Ile Val Pro Tyr Ile 610 615 620 Gly Leu Ala Leu Asn Val Gly Asn Glu Thr Ala Lys Gly Asn Phe Glu 625 630 635 640 Asn Ala Phe Glu Ile Ala Gly Ala Ser Ile Leu Leu Glu Phe Ile Pro 645 650 655 Glu Leu Leu Ile Pro Val Val Gly Ala Phe Leu Leu Glu Ser Tyr Ile 660 665 670 Asp Asn Lys Asn Lys Ile Ile Lys Thr Ile Asp Asn Ala Leu Thr Lys 675 680 685 Arg Asn Glu Lys Trp Ser Asp Met Tyr Gly Leu Ile Val Ala Gln Trp 690 695 700 Leu Ser Thr Val Asn Thr Gln Phe Tyr Thr Ile Lys Glu Gly Met Tyr 705 710 715 720 Lys Ala Leu Asn Tyr Gln Ala Gln Ala Leu Glu Glu Ile Ile Lys Tyr 725 730 735 Arg Tyr Asn Ile Tyr Ser Glu Lys Glu Lys Ser Asn Ile Asn Ile Asp 740 745 750 Phe Asn Asp Ile Asn Ser Lys Leu Asn Glu Gly Ile Asn Gln Ala Ile 755 760 765 Asp Asn Ile Asn Asn Phe Ile Asn Gly Cys Ser Val Ser Tyr Leu Met 770 775 780 Lys Lys Met Ile Pro Leu Ala Val Glu Lys Leu Leu Asp Phe Asp Asn 785 790 795 800 Thr Leu Lys Lys Asn Leu Leu Asn Tyr Ile Asp Glu Asn Lys Leu Tyr 805 810 815 Leu Ile Gly Ser Ala Glu Tyr Glu Lys Ser Lys Val Asn Lys Tyr Leu 820 825 830 Lys Thr Ile Met Pro Phe Asp Leu Ser Ile Tyr Thr Asn Asp Thr Ile 835 840 845 Leu Ile Glu Met Phe Asn Lys Tyr Asn Ser Glu Ile Leu Asn Asn Ile 850 855 860 Ile Leu Asn Leu Arg Tyr Lys Asp Asn Asn Leu Ile Asp Leu Ser Gly 865 870 875 880 Tyr Gly Ala Lys Val Glu Val Tyr Asp Gly Val Glu Leu Asn Asp Lys 885 890 895 Asn Gln Phe Lys Leu Thr Ser Ser Ala Asn Ser Lys Ile Arg Val Thr 900 905 910 Gln Asn Gln Asn Ile Ile Phe Asn Ser Val Phe Leu Asp Phe Ser Val 915 920 925 Ser Phe Trp Ile Arg Ile Pro Lys Tyr Lys Asn Asp Gly Ile Gln Asn 930 935 940 Tyr Ile His Asn Glu Tyr Thr Ile Ile Asn Cys Met Lys Asn Asn Ser 945 950 955 960 Gly Trp Lys Ile Ser Ile Arg Gly Asn Arg Ile Ile Trp Thr Leu Ile 965 970 975 Asp Ile Asn Gly Lys Thr Lys Ser Val Phe Phe Glu Tyr Asn Ile Arg 980 985 990 Glu Asp Ile Ser Glu Tyr Ile Asn Arg Trp Phe Phe Val Thr Ile Thr 995 1000 1005 Asn Asn Leu Asn Asn Ala Lys Ile Tyr Ile Asn Gly Lys Leu Glu Ser 1010 1015 1020 Asn Thr Asp Ile Lys Asp Ile Arg Glu Val Ile Ala Asn Gly Glu Ile 1025 1030 1035 1040 Ile Phe Lys Leu Asp Gly Asp Ile Asp Arg Thr Gln Phe Ile Trp Met 1045 1050 1055 Lys Tyr Phe Ser Ile Phe Asn Thr Glu Leu Ser Gln Ser Asn Ile Glu 1060 1065 1070 Glu Arg Tyr Lys Ile Gln Ser Tyr Ser Glu Tyr Leu Lys Asp Phe Trp 1075 1080 1085 Gly Asn Pro Leu Met Tyr Asn Lys Glu Tyr Tyr Met Phe Asn Ala Gly 1090 1095 1100 Asn Lys Asn Ser Tyr Ile Lys Leu Lys Lys Asp Ser Pro Val Gly Glu 1105 1110 1115 1120 Ile Leu Thr Arg Ser Lys Tyr Asn Gln Asn Ser Lys Tyr Ile Asn Tyr 1125 1130 1135 Arg Asp Leu Tyr Ile Gly Glu Lys Phe Ile Ile Arg Arg Lys Ser Asn 1140 1145 1150 Ser Gln Ser Ile Asn Asp Asp Ile Val Arg Lys Glu Asp Tyr Ile Tyr 1155 1160 1165 Leu Asp Phe Phe Asn Leu Asn Gln Glu Trp Arg Val Tyr Thr Tyr Lys 1170 1175 1180 Tyr Phe Lys Lys Glu Glu Glu Lys Leu Phe Leu Ala Pro Ile Ser Asp 1185 1190 1195 1200 Ser Asp Glu Phe Tyr Asn Thr Ile Gln Ile Lys Glu Tyr Asp Glu Gln 1205 1210 1215 Pro Thr Tyr Ser Cys Gln Leu Leu Phe Lys Lys Asp Glu Glu Ser Thr 1220 1225 1230 Asp Glu Ile Gly Leu Ile Gly Ile His Arg Phe Tyr Glu Ser Gly Ile 1235 1240 1245 Val Phe Glu Glu Tyr Lys Asp Tyr Phe Cys Ile Ser Lys Trp Tyr Leu 1250 1255 1260 Lys Glu Val Lys Arg Lys Pro Tyr Asn Leu Lys Leu Gly Cys Asn Trp 1265 1270 1275 1280 Gln Phe Ile Pro Lys Asp Glu Gly Trp Thr Glu 1285 1290

43 AGATCTCGAT CCCGCGAAAT TAATACGACT CACTATAGGG GAATTGTGAG CGGATAACAA 60 TTCCCCTCTA GAAATAATTT TGTTTAACTT TAAGAAGGAG ATATACC ATG GGC CAT 116 Met Gly His 1 CAT CAT CAT CAT CAT CAT CAT CAT CAC AGC AGC GGC CAT ATC GAA GGT 164 His His His His His His His His His Ser Ser Gly His Ile Glu Gly 5 10 15 CGT CAT ATG GCT AGC ATG GCT GAT ACA ATA CTA ATA GAA ATG TTT AAT 212 Arg His Met Ala Ser Met Ala Asp Thr Ile Leu Ile Glu Met Phe Asn 20 25 30 35 AAA TAT AAT AGC GAA ATT TTA AAT AAT ATT ATC TTA AAT TTA AGA TAT 260 Lys Tyr Asn Ser Glu Ile Leu Asn Asn Ile Ile Leu Asn Leu Arg Tyr 40 45 50 AGA GAT AAT AAT TTA ATA GAT TTA TCA GGA TAT GGA GCA AAG GTA GAG 308 Arg Asp Asn Asn Leu Ile Asp Leu Ser Gly Tyr Gly Ala Lys Val Glu 55 60 65 GTA TAT GAT GGG GTC AAG CTT AAT GAT AAA AAT CAA TTT AAA TTA ACT 356 Val Tyr Asp Gly Val Lys Leu Asn Asp Lys Asn Gln Phe Lys Leu Thr 70 75 80 AGT TCA GCA GAT AGT AAG ATT AGA GTC ACT CAA AAT CAG AAT ATT ATA 404 Ser Ser Ala Asp Ser Lys Ile Arg Val Thr Gln Asn Gln Asn Ile Ile 85 90 95 TTT AAT AGT ATG TTC CTT GAT TTT AGC GTT AGC TTT TGG ATA AGG ATA 452 Phe Asn Ser Met Phe Leu Asp Phe Ser Val Ser Phe Trp Ile Arg Ile 100 105 110 115 CCT AAA TAT AGG AAT GAT GAT ATA CAA AAT TAT ATT CAT AAT GAA TAT 500 Pro Lys Tyr Arg Asn Asp Asp Ile Gln Asn Tyr Ile His Asn Glu Tyr 120 125 130 ACG ATA ATT AAT TGT ATG AAA AAT AAT TCA GGC TGG AAA ATA TCT ATT 548 Thr Ile Ile Asn Cys Met Lys Asn Asn Ser Gly Trp Lys Ile Ser Ile 135 140 145 AGG GGT AAT AGG ATA ATA TGG ACC TTA ATT GAT ATA AAT GGA AAA ACC 596 Arg Gly Asn Arg Ile Ile Trp Thr Leu Ile Asp Ile Asn Gly Lys Thr 150 155 160 AAA TCA GTA TTT TTT GAA TAT AAC ATA AGA GAA GAT ATA TCA GAG TAT 644 Lys Ser Val Phe Phe Glu Tyr Asn Ile Arg Glu Asp Ile Ser Glu Tyr 165 170 175 ATA AAT AGA TGG TTT TTT GTA ACT ATT ACT AAT AAT TTG GAT AAT GCT 692 Ile Asn Arg Trp Phe Phe Val Thr Ile Thr Asn Asn Leu Asp Asn Ala 180 185 190 195 AAA ATT TAT ATT AAT GGC ACG TTA GAA TCA AAT ATG GAT ATT AAA GAT 740 Lys Ile Tyr Ile Asn Gly Thr Leu Glu Ser Asn Met Asp Ile Lys Asp 200 205 210 ATA GGA GAA GTT ATT GTT AAT GGT GAA ATA ACA TTT AAA TTA GAT GGT 788 Ile Gly Glu Val Ile Val Asn Gly Glu Ile Thr Phe Lys Leu Asp Gly 215 220 225 GAT GTA GAT AGA ACA CAA TTT ATT TGG ATG AAA TAT TTT AGT ATT TTT 836 Asp Val Asp Arg Thr Gln Phe Ile Trp Met Lys Tyr Phe Ser Ile Phe 230 235 240 AAT ACG CAA TTA AAT CAA TCA AAT ATT AAA GAG ATA TAT AAA ATT CAA 884 Asn Thr Gln Leu Asn Gln Ser Asn Ile Lys Glu Ile Tyr Lys Ile Gln 245 250 255 TCA TAT AGC GAA TAC TTA AAA GAT TTT TGG GGA AAT CCT TTA ATG TAT 932 Ser Tyr Ser Glu Tyr Leu Lys Asp Phe Trp Gly Asn Pro Leu Met Tyr 260 265 270 275 AAT AAA GAA TAT TAT ATG TTT AAT GCG GGG AAT AAA AAT TCA TAT ATT 980 Asn Lys Glu Tyr Tyr Met Phe Asn Ala Gly Asn Lys Asn Ser Tyr Ile 280 285 290 AAA CTA GTG AAA GAT TCA TCT GTA GGT GAA ATA TTA ATA CGT AGC AAA 1028 Lys Leu Val Lys Asp Ser Ser Val Gly Glu Ile Leu Ile Arg Ser Lys 295 300 305 TAT AAT CAG AAT TCC AAT TAT ATA AAT TAT AGA AAT TTA TAT ATT GGA 1076 Tyr Asn Gln Asn Ser Asn Tyr Ile Asn Tyr Arg Asn Leu Tyr Ile Gly 310 315 320 GAA AAA TTT ATT ATA AGA AGA GAG TCA AAT TCT CAA TCT ATA AAT GAT 1124 Glu Lys Phe Ile Ile Arg Arg Glu Ser Asn Ser Gln Ser Ile Asn Asp 325 330 335 GAT ATA GTT AGA AAA GAA GAT TAT ATA CAT CTA GAT TTG GTA CTT CAC 1172 Asp Ile Val Arg Lys Glu Asp Tyr Ile His Leu Asp Leu Val Leu His 340 345 350 355 CAT GAA GAG TGG AGA GTA TAT GCC TAT AAA TAT TTT AAG GAA CAG GAA 1220 His Glu Glu Trp Arg Val Tyr Ala Tyr Lys Tyr Phe Lys Glu Gln Glu 360 365 370 GAA AAA TTG TTT TTA TCT ATT ATA AGT GAT TCT AAT GAA TTT TAT AAG 1268 Glu Lys Leu Phe Leu Ser Ile Ile Ser Asp Ser Asn Glu Phe Tyr Lys 375 380 385 ACT ATA GAA ATA AAA GAA TAT GAT GAA CAG CCA TCA TAT AGT TGT CAG 1316 Thr Ile Glu Ile Lys Glu Tyr Asp Glu Gln Pro Ser Tyr Ser Cys Gln 390 395 400 TTG CTT TTT AAA AAA GAT GAA GAA AGT ACT GAT GAT ATA GGA TTG ATT 1364 Leu Leu Phe Lys Lys Asp Glu Glu Ser Thr Asp Asp Ile Gly Leu Ile 405 410 415 GGT ATT CAT CGT TTC TAC GAA TCT GGA GTT TTA CGT AAA AAG TAT AAA 1412 Gly Ile His Arg Phe Tyr Glu Ser Gly Val Leu Arg Lys Lys Tyr Lys 420 425 430 435 GAT TAT TTT TGT ATA AGT AAA TGG TAC TTA AAA GAG GTA AAA AGG AAA 1460 Asp Tyr Phe Cys Ile Ser Lys Trp Tyr Leu Lys Glu Val Lys Arg Lys 440 445 450 CCA TAT AAG TCA AAT TTG GGA TGT AAT TGG CAG TTT ATT CCT AAA GAT 1508 Pro Tyr Lys Ser Asn Leu Gly Cys Asn Trp Gln Phe Ile Pro Lys Asp 455 460 465 GAA GGG TGG ACT GAA TAA 1526 Glu Gly Trp Thr Glu 470

44 Met Gly His His His His His His His His His His Ser Ser Gly His 1 5 10 15 Ile Glu Gly Arg His Met Ala Ser Met Ala Asp Thr Ile Leu Ile Glu 20 25 30 Met Phe Asn Lys Tyr Asn Ser Glu Ile Leu Asn Asn Ile Ile Leu Asn 35 40 45 Leu Arg Tyr Arg Asp Asn Asn Leu Ile Asp Leu Ser Gly Tyr Gly Ala 50 55 60 Lys Val Glu Val Tyr Asp Gly Val Lys Leu Asn Asp Lys Asn Gln Phe 65 70 75 80 Lys Leu Thr Ser Ser Ala Asp Ser Lys Ile Arg Val Thr Gln Asn Gln 85 90 95 Asn Ile Ile Phe Asn Ser Met Phe Leu Asp Phe Ser Val Ser Phe Trp 100 105 110 Ile Arg Ile Pro Lys Tyr Arg Asn Asp Asp Ile Gln Asn Tyr Ile His 115 120 125 Asn Glu Tyr Thr Ile Ile Asn Cys Met Lys Asn Asn Ser Gly Trp Lys 130 135 140 Ile Ser Ile Arg Gly Asn Arg Ile Ile Trp Thr Leu Ile Asp Ile Asn 145 150 155 160 Gly Lys Thr Lys Ser Val Phe Phe Glu Tyr Asn Ile Arg Glu Asp Ile 165 170 175 Ser Glu Tyr Ile Asn Arg Trp Phe Phe Val Thr Ile Thr Asn Asn Leu 180 185 190 Asp Asn Ala Lys Ile Tyr Ile Asn Gly Thr Leu Glu Ser Asn Met Asp 195 200 205 Ile Lys Asp Ile Gly Glu Val Ile Val Asn Gly Glu Ile Thr Phe Lys 210 215 220 Leu Asp Gly Asp Val Asp Arg Thr Gln Phe Ile Trp Met Lys Tyr Phe 225 230 235 240 Ser Ile Phe Asn Thr Gln Leu Asn Gln Ser Asn Ile Lys Glu Ile Tyr 245 250 255 Lys Ile Gln Ser Tyr Ser Glu Tyr Leu Lys Asp Phe Trp Gly Asn Pro 260 265 270 Leu Met Tyr Asn Lys Glu Tyr Tyr Met Phe Asn Ala Gly Asn Lys Asn 275 280 285 Ser Tyr Ile Lys Leu Val Lys Asp Ser Ser Val Gly Glu Ile Leu Ile 290 295 300 Arg Ser Lys Tyr Asn Gln Asn Ser Asn Tyr Ile Asn Tyr Arg Asn Leu 305 310 315 320 Tyr Ile Gly Glu Lys Phe Ile Ile Arg Arg Glu Ser Asn Ser Gln Ser 325 330 335 Ile Asn Asp Asp Ile Val Arg Lys Glu Asp Tyr Ile His Leu Asp Leu 340 345 350 Val Leu His His Glu Glu Trp Arg Val Tyr Ala Tyr Lys Tyr Phe Lys 355 360 365 Glu Gln Glu Glu Lys Leu Phe Leu Ser Ile Ile Ser Asp Ser Asn Glu 370 375 380 Phe Tyr Lys Thr Ile Glu Ile Lys Glu Tyr Asp Glu Gln Pro Ser Tyr 385 390 395 400 Ser Cys Gln Leu Leu Phe Lys Lys Asp Glu Glu Ser Thr Asp Asp Ile 405 410 415 Gly Leu Ile Gly Ile His Arg Phe Tyr Glu Ser Gly Val Leu Arg Lys 420 425 430 Lys Tyr Lys Asp Tyr Phe Cys Ile Ser Lys Trp Tyr Leu Lys Glu Val 435 440 445 Lys Arg Lys Pro Tyr Lys Ser Asn Leu Gly Cys Asn Trp Gln Phe Ile 450 455 460 Pro Lys Asp Glu Gly Trp Thr Glu 465 470

45 AGATCTCGAT CCCGCGAAAT TAATACGACT CACTATAGGG GAATTGTGAG CGGATAACAA 60 TTCCCCTCTA GAAATAATTT TGTTTAACTT TAAGAAGGAG ATATACC ATG GGC CAT 116 Met Gly His 1 CAT CAT CAT CAT CAT CAT CAT CAT CAC AGC AGC GGC CAT ATC GAA GGT 164 His His His His His His His His His Ser Ser Gly His Ile Glu Gly 5 10 15 CGT CAT ATG GCT AGC ATG GCT GAT ACA ATA CTA ATA GAA ATG TTT AAT 212 Arg His Met Ala Ser Met Ala Asp Thr Ile Leu Ile Glu Met Phe Asn 20 25 30 35 AAA TAT AAT AGC GAA ATT TTA AAT AAT ATT ATC TTA AAT TTA AGA TAT 260 Lys Tyr Asn Ser Glu Ile Leu Asn Asn Ile Ile Leu Asn Leu Arg Tyr 40 45 50 AAG GAT AAT AAT TTA ATA GAT TTA TCA GGA TAT GGG GCA AAG GTA GAG 308 Lys Asp Asn Asn Leu Ile Asp Leu Ser Gly Tyr Gly Ala Lys Val Glu 55 60 65 GTA TAT GAT GGA GTC GAG CTT AAT GAT AAA AAT CAA TTT AAA TTA ACT 356 Val Tyr Asp Gly Val Glu Leu Asn Asp Lys Asn Gln Phe Lys Leu Thr 70 75 80 AGT TCA GCA AAT AGT AAG ATT AGA GTG ACT CAA AAT CAG AAT ATC ATA 404 Ser Ser Ala Asn Ser Lys Ile Arg Val Thr Gln Asn Gln Asn Ile Ile 85 90 95 TTT AAT AGT GTG TTC CTT GAT TTT AGC GTT AGC TTT TGG ATA AGA ATA 452 Phe Asn Ser Val Phe Leu Asp Phe Ser Val Ser Phe Trp Ile Arg Ile 100 105 110 115 CCT AAA TAT AAG AAT GAT GGT ATA CAA AAT TAT ATT CAT AAT GAA TAT 500 Pro Lys Tyr Lys Asn Asp Gly Ile Gln Asn Tyr Ile His Asn Glu Tyr 120 125 130 ACA ATA ATT AAT TGT ATG AAA AAT AAT TCG GGC TGG AAA ATA TCT ATT 548 Thr Ile Ile Asn Cys Met Lys Asn Asn Ser Gly Trp Lys Ile Ser Ile 135 140 145 AGG GGT AAT AGG ATA ATA TGG ACT TTA ATT GAT ATA AAT GGA AAA ACC 596 Arg Gly Asn Arg Ile Ile Trp Thr Leu Ile Asp Ile Asn Gly Lys Thr 150 155 160 AAA TCG GTA TTT TTT GAA TAT AAC ATA AGA GAA GAT ATA TCA GAG TAT 644 Lys Ser Val Phe Phe Glu Tyr Asn Ile Arg Glu Asp Ile Ser Glu Tyr 165 170 175 ATA AAT AGA TGG TTT TTT GTA ACT ATT ACT AAT AAT TTG AAT AAC GCT 692 Ile Asn Arg Trp Phe Phe Val Thr Ile Thr Asn Asn Leu Asn Asn Ala 180 185 190 195 AAA ATT TAT ATT AAT GGT AAG CTA GAA TCA AAT ACA GAT ATT AAA GAT 740 Lys Ile Tyr Ile Asn Gly Lys Leu Glu Ser Asn Thr Asp Ile Lys Asp 200 205 210 ATA AGA GAA GTT ATT GCT AAT GGT GAA ATA ATA TTT AAA TTA GAT GGT 788 Ile Arg Glu Val Ile Ala Asn Gly Glu Ile Ile Phe Lys Leu Asp Gly 215 220 225 GAT ATA GAT AGA ACA CAA TTT ATT TGG ATG AAA TAT TTC AGT ATT TTT 836 Asp Ile Asp Arg Thr Gln Phe Ile Trp Met Lys Tyr Phe Ser Ile Phe 230 235 240 AAT ACG GAA TTA AGT CAA TCA AAT ATT GAA GAA AGA TAT AAA ATT CAA 884 Asn Thr Glu Leu Ser Gln Ser Asn Ile Glu Glu Arg Tyr Lys Ile Gln 245 250 255 TCA TAT AGC GAA TAT TTA AAA GAT TTT TGG GGA AAT CCT TTA ATG TAC 932 Ser Tyr Ser Glu Tyr Leu Lys Asp Phe Trp Gly Asn Pro Leu Met Tyr 260 265 270 275 AAT AAA GAA TAT TAT ATG TTT AAT GCG GGG AAT AAA AAT TCA TAT ATT 980 Asn Lys Glu Tyr Tyr Met Phe Asn Ala Gly Asn Lys Asn Ser Tyr Ile 280 285 290 AAA CTA AAG AAA GAT TCA CCT GTA GGT GAA ATT TTA ACA CGT AGC AAA 1028 Lys Leu Lys Lys Asp Ser Pro Val Gly Glu Ile Leu Thr Arg Ser Lys 295 300 305 TAT AAT CAA AAT TCT AAA TAT ATA AAT TAT AGA GAT TTA TAT ATT GGA 1076 Tyr Asn Gln Asn Ser Lys Tyr Ile Asn Tyr Arg Asp Leu Tyr Ile Gly 310 315 320 GAA AAA TTT ATT ATA AGA AGA AAG TCA AAT TCT CAA TCT ATA AAT GAT 1124 Glu Lys Phe Ile Ile Arg Arg Lys Ser Asn Ser Gln Ser Ile Asn Asp 325 330 335 GAT ATA GTT AGA AAA GAA GAT TAT ATA TAT CTA GAT TTT TTT AAT TTA 1172 Asp Ile Val Arg Lys Glu Asp Tyr Ile Tyr Leu Asp Phe Phe Asn Leu 340 345 350 355 AAT CAA GAG TGG AGA GTA TAT ACC TAT AAA TAT TTT AAG AAA GAG GAA 1220 Asn Gln Glu Trp Arg Val Tyr Thr Tyr Lys Tyr Phe Lys Lys Glu Glu 360 365 370 GAA AAA TTG TTT TTA GCT CCT ATA AGT GAT TCT GAT GAG TTT TAC AAT 1268 Glu Lys Leu Phe Leu Ala Pro Ile Ser Asp Ser Asp Glu Phe Tyr Asn 375 380 385 ACT ATA CAA ATA AAA GAA TAT GAT GAA CAG CCA ACA TAT AGT TGT CAG 1316 Thr Ile Gln Ile Lys Glu Tyr Asp Glu Gln Pro Thr Tyr Ser Cys Gln 390 395 400 TTG CTT TTT AAA AAA GAT GAA GAA AGT ACT GAT GAG ATA GGA TTG ATT 1364 Leu Leu Phe Lys Lys Asp Glu Glu Ser Thr Asp Glu Ile Gly Leu Ile 405 410 415 GGT ATT CAT CGT TTC TAC GAA TCT GGA ATT GTA TTT GAA GAG TAT AAA 1412 Gly Ile His Arg Phe Tyr Glu Ser Gly Ile Val Phe Glu Glu Tyr Lys 420 425 430 435 GAT TAT TTT TGT ATA AGT AAA TGG TAC TTA AAA GAG GTA AAA AGG AAA 1460 Asp Tyr Phe Cys Ile Ser Lys Trp Tyr Leu Lys Glu Val Lys Arg Lys 440 445 450 CCA TAT AAT TTA AAA TTG GGA TGT AAT TGG CAG TTT ATT CCT AAA GAT 1508 Pro Tyr Asn Leu Lys Leu Gly Cys Asn Trp Gln Phe Ile Pro Lys Asp 455 460 465 GAA GGG TGG ACT GAA TAAAAGCTTG CGGCCGCACT CGAG 1547 Glu Gly Trp Thr Glu 470

46 Met Gly His His His His His His His His His His Ser Ser Gly His 1 5 10 15 Ile Glu Gly Arg His Met Ala Ser Met Ala Asp Thr Ile Leu Ile Glu 20 25 30 Met Phe Asn Lys Tyr Asn Ser Glu Ile Leu Asn Asn Ile Ile Leu Asn 35 40 45 Leu Arg Tyr Lys Asp Asn Asn Leu Ile Asp Leu Ser Gly Tyr Gly Ala 50 55 60 Lys Val Glu Val Tyr Asp Gly Val Glu Leu Asn Asp Lys Asn Gln Phe 65 70 75 80 Lys Leu Thr Ser Ser Ala Asn Ser Lys Ile Arg Val Thr Gln Asn Gln 85 90 95 Asn Ile Ile Phe Asn Ser Val Phe Leu Asp Phe Ser Val Ser Phe Trp 100 105 110 Ile Arg Ile Pro Lys Tyr Lys Asn Asp Gly Ile Gln Asn Tyr Ile His 115 120 125 Asn Glu Tyr Thr Ile Ile Asn Cys Met Lys Asn Asn Ser Gly Trp Lys 130 135 140 Ile Ser Ile Arg Gly Asn Arg Ile Ile Trp Thr Leu Ile Asp Ile Asn 145 150 155 160 Gly Lys Thr Lys Ser Val Phe Phe Glu Tyr Asn Ile Arg Glu Asp Ile 165 170 175 Ser Glu Tyr Ile Asn Arg Trp Phe Phe Val Thr Ile Thr Asn Asn Leu 180 185 190 Asn Asn Ala Lys Ile Tyr Ile Asn Gly Lys Leu Glu Ser Asn Thr Asp 195 200 205 Ile Lys Asp Ile Arg Glu Val Ile Ala Asn Gly Glu Ile Ile Phe Lys 210 215 220 Leu Asp Gly Asp Ile Asp Arg Thr Gln Phe Ile Trp Met Lys Tyr Phe 225 230 235 240 Ser Ile Phe Asn Thr Glu Leu Ser Gln Ser Asn Ile Glu Glu Arg Tyr 245 250 255 Lys Ile Gln Ser Tyr Ser Glu Tyr Leu Lys Asp Phe Trp Gly Asn Pro 260 265 270 Leu Met Tyr Asn Lys Glu Tyr Tyr Met Phe Asn Ala Gly Asn Lys Asn 275 280 285 Ser Tyr Ile Lys Leu Lys Lys Asp Ser Pro Val Gly Glu Ile Leu Thr 290 295 300 Arg Ser Lys Tyr Asn Gln Asn Ser Lys Tyr Ile Asn Tyr Arg Asp Leu 305 310 315 320 Tyr Ile Gly Glu Lys Phe Ile Ile Arg Arg Lys Ser Asn Ser Gln Ser 325 330 335 Ile Asn Asp Asp Ile Val Arg Lys Glu Asp Tyr Ile Tyr Leu Asp Phe 340 345 350 Phe Asn Leu Asn Gln Glu Trp Arg Val Tyr Thr Tyr Lys Tyr Phe Lys 355 360 365 Lys Glu Glu Glu Lys Leu Phe Leu Ala Pro Ile Ser Asp Ser Asp Glu 370 375 380 Phe Tyr Asn Thr Ile Gln Ile Lys Glu Tyr Asp Glu Gln Pro Thr Tyr 385 390 395 400 Ser Cys Gln Leu Leu Phe Lys Lys Asp Glu Glu Ser Thr Asp Glu Ile 405 410 415 Gly Leu Ile Gly Ile His Arg Phe Tyr Glu Ser Gly Ile Val Phe Glu 420 425 430 Glu Tyr Lys Asp Tyr Phe Cys Ile Ser Lys Trp Tyr Leu Lys Glu Val 435 440 445 Lys Arg Lys Pro Tyr Asn Leu Lys Leu Gly Cys Asn Trp Gln Phe Ile 450 455 460 Pro Lys Asp Glu Gly Trp Thr Glu 465 470

47 CGCCATGGCT GATACAATAC TAATAGAAAT G 31

48 GCAAGCTTTT ATTCAGTCCA CCCTTCATC 29

49 ATG CCA ACA ATT AAT AGT TTT AAT TAT AAT GAT CCT GTT AAT AAT AGA 48 Met Pro Thr Ile Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asn Arg 1 5 10 15 ACA ATT TTA TAT ATT AAA CCA GGC GGT TGT CAA CAA TTT TAT AAA TCA 96 Thr Ile Leu Tyr Ile Lys Pro Gly Gly Cys Gln Gln Phe Tyr Lys Ser 20 25 30 TTT AAT ATT ATG AAA AAT ATT TGG ATA ATT CCA GAG AGA AAT GTA ATT 144 Phe Asn Ile Met Lys Asn Ile Trp Ile Ile Pro Glu Arg Asn Val Ile 35 40 45 GGT ACA ATT CCC CAA GAT TTT CTT CCG CCT ACT TCA TTG AAA AAT GGA 192 Gly Thr Ile Pro Gln Asp Phe Leu Pro Pro Thr Ser Leu Lys Asn Gly 50 55 60 GAT AGT AGT TAT TAT GAC CCT AAT TAT TTA CAA AGT GAT CAA GAA AAG 240 Asp Ser Ser Tyr Tyr Asp Pro Asn Tyr Leu Gln Ser Asp Gln Glu Lys 65 70 75 80 GAT AAA TTT TTA AAA ATA GTC ACA AAA ATA TTT AAT AGA ATA AAT GAT 288 Asp Lys Phe Leu Lys Ile Val Thr Lys Ile Phe Asn Arg Ile Asn Asp 85 90 95 AAT CTT TCA GGA AGG ATT TTA TTA GAA GAA CTG TCA AAA GCT AAT CCA 336 Asn Leu Ser Gly Arg Ile Leu Leu Glu Glu Leu Ser Lys Ala Asn Pro 100 105 110 TAT TTA GGA AAT GAT AAT ACT CCA GAT GGT GAC TTC ATT ATT AAT GAT 384 Tyr Leu Gly Asn Asp Asn Thr Pro Asp Gly Asp Phe Ile Ile Asn Asp 115 120 125 GCA TCA GCA GTT CCA ATT CAA TTC TCA AAT GGT AGC CAA AGC ATA CTA 432 Ala Ser Ala Val Pro Ile Gln Phe Ser Asn Gly Ser Gln Ser Ile Leu 130 135 140 TTA CCT AAT GTT ATT ATA ATG GGA GCA GAG CCT GAT TTA TTT GAA ACT 480 Leu Pro Asn Val Ile Ile Met Gly Ala Glu Pro Asp Leu Phe Glu Thr 145 150 155 160 AAC AGT TCC AAT ATT TCT CTA AGA AAT AAT TAT ATG CCA AGC AAT CAC 528 Asn Ser Ser Asn Ile Ser Leu Arg Asn Asn Tyr Met Pro Ser Asn His 165 170 175 GGT TTT GGA TCA ATA GCT ATA GTA ACA TTC TCA CCT GAA TAT TCT TTT 576 Gly Phe Gly Ser Ile Ala Ile Val Thr Phe Ser Pro Glu Tyr Ser Phe 180 185 190 AGA TTT AAA GAT AAT AGT ATG AAT GAA TTT ATT CAA GAT CCT GCT CTT 624 Arg Phe Lys Asp Asn Ser Met Asn Glu Phe Ile Gln Asp Pro Ala Leu 195 200 205 ACA TTA ATG CAT GAA TTA ATA CAT TCA TTA CAT GGA CTA TAT GGG GCT 672 Thr Leu Met His Glu Leu Ile His Ser Leu His Gly Leu Tyr Gly Ala 210 215 220 AAA GGG ATT ACT ACA AAG TAT ACT ATA ACA CAA AAA CAA AAT CCC CTA 720 Lys Gly Ile Thr Thr Lys Tyr Thr Ile Thr Gln Lys Gln Asn Pro Leu 225 230 235 240 ATA ACA AAT ATA AGA GGT ACA AAT ATT GAA GAA TTC TTA ACT TTT GGA 768 Ile Thr Asn Ile Arg Gly Thr Asn Ile Glu Glu Phe Leu Thr Phe Gly 245 250 255 GGT ACT GAT TTA AAC ATT ATT ACT AGT GCT CAG TCC AAT GAT ATC TAT 816 Gly Thr Asp Leu Asn Ile Ile Thr Ser Ala Gln Ser Asn Asp Ile Tyr 260 265 270 ACT AAT CTT CTA GCT GAT TAT AAA AAA ATA GCG TCT AAA CTT AGC AAA 864 Thr Asn Leu Leu Ala Asp Tyr Lys Lys Ile Ala Ser Lys Leu Ser Lys 275 280 285 GTA CAA GTA TCT AAT CCA CTA CTT AAT CCT TAT AAA GAT GTT TTT GAA 912 Val Gln Val Ser Asn Pro Leu Leu Asn Pro Tyr Lys Asp Val Phe Glu 290 295 300 GCA AAG TAT GGA TTA GAT AAA GAT GCT AGC GGA ATT TAT TCG GTA AAT 960 Ala Lys Tyr Gly Leu Asp Lys Asp Ala Ser Gly Ile Tyr Ser Val Asn 305 310 315 320 ATA AAC AAA TTT AAT GAT ATT TTT AAA AAA TTA TAC AGC TTT ACG GAA 1008 Ile Asn Lys Phe Asn Asp Ile Phe Lys Lys Leu Tyr Ser Phe Thr Glu 325 330 335 TTT GAT TTA GCA ACT AAA TTT CAA GTT AAA TGT AGG CAA ACT TAT ATT 1056 Phe Asp Leu Ala Thr Lys Phe Gln Val Lys Cys Arg Gln Thr Tyr Ile 340 345 350 GGA CAG TAT AAA TAC TTC AAA CTT TCA AAC TTG TTA AAT GAT TCT ATT 1104 Gly Gln Tyr Lys Tyr Phe Lys Leu Ser Asn Leu Leu Asn Asp Ser Ile 355 360 365 TAT AAT ATA TCA GAA GGC TAT AAT ATA AAT AAT TTA AAG GTA AAT TTT 1152 Tyr Asn Ile Ser Glu Gly Tyr Asn Ile Asn Asn Leu Lys Val Asn Phe 370 375 380 AGA GGA CAG AAT GCA AAT TTA AAT CCT AGA ATT ATT ACA CCA ATT ACA 1200 Arg Gly Gln Asn Ala Asn Leu Asn Pro Arg Ile Ile Thr Pro Ile Thr 385 390 395 400 GGT AGA GGA CTA GTA AAA AAA ATC ATT AGA TTT TGT AAA AAT ATT GTT 1248 Gly Arg Gly Leu Val Lys Lys Ile Ile Arg Phe Cys Lys Asn Ile Val 405 410 415 TCT GTA AAA GGC ATA AGG AAA TCA ATA TGT ATC GAA ATA AAT AAT GGT 1296 Ser Val Lys Gly Ile Arg Lys Ser Ile Cys Ile Glu Ile Asn Asn Gly 420 425 430 GAG TTA TTT TTT GTG GCT TCC GAG AAT AGT TAT AAT GAT GAT AAT ATA 1344 Glu Leu Phe Phe Val Ala Ser Glu Asn Ser Tyr Asn Asp Asp Asn Ile 435 440 445 AAT ACT CCT AAA GAA ATT GAC GAT ACA GTA ACT TCA AAT AAT AAT TAT 1392 Asn Thr Pro Lys Glu Ile Asp Asp Thr Val Thr Ser Asn Asn Asn Tyr 450 455 460 GAA AAT GAT TTA GAT CAG GTT ATT TTA AAT TTT AAT AGT GAA TCA GCA 1440 Glu Asn Asp Leu Asp Gln Val Ile Leu Asn Phe Asn Ser Glu Ser Ala 465 470 475 480 CCT GGA CTT TCA GAT GAA AAA TTA AAT TTA ACT ATC CAA AAT GAT GCT 1488 Pro Gly Leu Ser Asp Glu Lys Leu Asn Leu Thr Ile Gln Asn Asp Ala 485 490 495 TAT ATA CCA AAA TAT GAT TCT AAT GGA ACA AGT GAT ATA GAA CAA CAT 1536 Tyr Ile Pro Lys Tyr Asp Ser Asn Gly Thr Ser Asp Ile Glu Gln His 500 505 510 GAT GTT AAT GAA CTT AAT GTA TTT TTC TAT TTA GAT GCA CAG AAA GTG 1584 Asp Val Asn Glu Leu Asn Val Phe Phe Tyr Leu Asp Ala Gln Lys Val 515 520 525 CCC GAA GGT GAA AAT AAT GTC AAT CTC ACC TCT TCA ATT GAT ACA GCA 1632 Pro Glu Gly Glu Asn Asn Val Asn Leu Thr Ser Ser Ile Asp Thr Ala 530 535 540 TTA TTA GAA CAA CCT AAA ATA TAT ACA TTT TTT TCA TCA GAA TTT ATT 1680 Leu Leu Glu Gln Pro Lys Ile Tyr Thr Phe Phe Ser Ser Glu Phe Ile 545 550 555 560 AAT AAT GTC AAT AAA CCT GTG CAA GCA GCA TTA TTT GTA AGC TGG ATA 1728 Asn Asn Val Asn Lys Pro Val Gln Ala Ala Leu Phe Val Ser Trp Ile 565 570 575 CAA CAA GTA TTA GTA GAT TTT ACT ACT GAA GCT AAC CAA AAA AGT ACT 1776 Gln Gln Val Leu Val Asp Phe Thr Thr Glu Ala Asn Gln Lys Ser Thr 580 585 590 GTT GAT AAA ATT GCA GAT ATT TCT ATA GTT GTT CCA TAT ATA GGT CTT 1824 Val Asp Lys Ile Ala Asp Ile Ser Ile Val Val Pro Tyr Ile Gly Leu 595 600 605 GCT TTA AAT ATA GGA AAT GAA GCA CAA AAA GGA AAT TTT AAA GAT GCA 1872 Ala Leu Asn Ile Gly Asn Glu Ala Gln Lys Gly Asn Phe Lys Asp Ala 610 615 620 CTT GAA TTA TTA GGA GCA GGT ATT TTA TTA GAA TTT GAA CCC GAG CTT 1920 Leu Glu Leu Leu Gly Ala Gly Ile Leu Leu Glu Phe Glu Pro Glu Leu 625 630 635 640 TTA ATT CCT ACA ATT TTA GTA TTC ACG ATA AAA TCT TTT TTA GGT TCA 1968 Leu Ile Pro Thr Ile Leu Val Phe Thr Ile Lys Ser Phe Leu Gly Ser 645 650 655 TCT GAT AAT AAA AAT AAA GTT ATT AAA GCA ATA AAT AAT GCA TTG AAA 2016 Ser Asp Asn Lys Asn Lys Val Ile Lys Ala Ile Asn Asn Ala Leu Lys 660 665 670 GAA AGA GAT GAA AAA TGG AAA GAA GTA TAT AGT TTT ATA GTA TCG AAT 2064 Glu Arg Asp Glu Lys Trp Lys Glu Val Tyr Ser Phe Ile Val Ser Asn 675 680 685 TGG ATG ACT AAA ATT AAT ACA CAA TTT AAT AAA AGA AAA GAA CAA ATG 2112 Trp Met Thr Lys Ile Asn Thr Gln Phe Asn Lys Arg Lys Glu Gln Met 690 695 700 TAT CAA GCT TTA CAA AAT CAA GTA AAT GCA CTT AAA GCA ATA ATA GAA 2160 Tyr Gln Ala Leu Gln Asn Gln Val Asn Ala Leu Lys Ala Ile Ile Glu 705 710 715 720 TCT AAG TAT AAT AGT TAT ACT TTA GAA GAA AAA AAT GAG CTT ACA AAT 2208 Ser Lys Tyr Asn Ser Tyr Thr Leu Glu Glu Lys Asn Glu Leu Thr Asn 725 730 735 AAA TAT GAT ATT GAG CAA ATA GAA AAT GAA CTT AAT CAA AAG GTT TCT 2256 Lys Tyr Asp Ile Glu Gln Ile Glu Asn Glu Leu Asn Gln Lys Val Ser 740 745 750 ATA GCA ATG AAT AAT ATA GAC AGG TTC TTA ACT GAA AGT TCT ATA TCT 2304 Ile Ala Met Asn Asn Ile Asp Arg Phe Leu Thr Glu Ser Ser Ile Ser 755 760 765 TAT TTA ATG AAA TTA ATA AAT GAA GTA AAA ATT AAT AAA TTA AGA GAA 2352 Tyr Leu Met Lys Leu Ile Asn Glu Val Lys Ile Asn Lys Leu Arg Glu 770 775 780 TAT GAT GAA AAT GTT AAA ACG TAT TTA TTA GAT TAT ATT ATA AAA CAT 2400 Tyr Asp Glu Asn Val Lys Thr Tyr Leu Leu Asp Tyr Ile Ile Lys His 785 790 795 800 GGA TCA ATC TTG GGA GAG AGT CAG CAA GAA CTA AAT TCT ATG GTA ATT 2448 Gly Ser Ile Leu Gly Glu Ser Gln Gln Glu Leu Asn Ser Met Val Ile 805 810 815 GAT ACC CTA AAT AAT AGT ATT CCT TTT AAG CTT TCT TCT TAT ACA GAT 2496 Asp Thr Leu Asn Asn Ser Ile Pro Phe Lys Leu Ser Ser Tyr Thr Asp 820 825 830 GAT AAA ATT TTA ATT TCA TAT TTT AAT AAG TTC TTT AAG AGA ATT AAA 2544 Asp Lys Ile Leu Ile Ser Tyr Phe Asn Lys Phe Phe Lys Arg Ile Lys 835 840 845 AGT AGT TCT GTT TTA AAT ATG AGA TAT AAA AAT GAT AAA TAC GTA GAT 2592 Ser Ser Ser Val Leu Asn Met Arg Tyr Lys Asn Asp Lys Tyr Val Asp 850 855 860 ACT TCA GGA TAT GAT TCA AAT ATA AAT ATT AAT GGA GAT GTA TAT AAA 2640 Thr Ser Gly Tyr Asp Ser Asn Ile Asn Ile Asn Gly Asp Val Tyr Lys 865 870 875 880 TAT CCA ACT AAT AAA AAT CAA TTT GGA ATA TAT AAT GAT AAA CTT AGT 2688 Tyr Pro Thr Asn Lys Asn Gln Phe Gly Ile Tyr Asn Asp Lys Leu Ser 885 890 895 GAA GTT AAT ATA TCT CAA AAT GAT TAC ATT ATA TAT GAT AAT AAA TAT 2736 Glu Val Asn Ile Ser Gln Asn Asp Tyr Ile Ile Tyr Asp Asn Lys Tyr 900 905 910 AAA AAT TTT AGT ATT AGT TTT TGG GTA AGA ATT CCT AAC TAT GAT AAT 2784 Lys Asn Phe Ser Ile Ser Phe Trp Val Arg Ile Pro Asn Tyr Asp Asn 915 920 925 AAG ATA GTA AAT GTT AAT AAT GAA TAC ACT ATA ATA AAT TGT ATG AGG 2832 Lys Ile Val Asn Val Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Arg 930 935 940 GAT AAT AAT TCA GGA TGG AAA GTA TCT CTT AAT CAT AAT GAA ATA ATT 2880 Asp Asn Asn Ser Gly Trp Lys Val Ser Leu Asn His Asn Glu Ile Ile 945 950 955 960 TGG ACA TTG CAA GAT AAT TCA GGA ATT AAT CAA AAA TTA GCA TTT AAC 2928 Trp Thr Leu Gln Asp Asn Ser Gly Ile Asn Gln Lys Leu Ala Phe Asn 965 970 975 TAT GGT AAC GCA AAT GGT ATT TCT GAT TAT ATA AAT AAG TGG ATT TTT 2976 Tyr Gly Asn Ala Asn Gly Ile Ser Asp Tyr Ile Asn Lys Trp Ile Phe 980 985 990 GTA ACT ATA ACT AAT GAT AGA TTA GGA GAT TCT AAA CTT TAT ATT AAT 3024 Val Thr Ile Thr Asn Asp Arg Leu Gly Asp Ser Lys Leu Tyr Ile Asn 995 1000 1005 GGA AAT TTA ATA GAT AAA AAA TCA ATT TTA AAT TTA GGT AAT ATT CAT 3072 Gly Asn Leu Ile Asp Lys Lys Ser Ile Leu Asn Leu Gly Asn Ile His 1010 1015 1020 GTT AGT GAC AAT ATA TTA TTT AAA ATA GTT AAT TGT AGT TAT ACA AGA 3120 Val Ser Asp Asn Ile Leu Phe Lys Ile Val Asn Cys Ser Tyr Thr Arg 1025 1030 1035 1040 TAT ATT GGT ATT AGA TAT TTT AAT ATT TTT GAT AAA GAA TTA GAT GAA 3168 Tyr Ile Gly Ile Arg Tyr Phe Asn Ile Phe Asp Lys Glu Leu Asp Glu 1045 1050 1055 ACA GAA ATT CAA ACT TTA TAT AAC AAT GAA CCT AAT GCA AAT ATT TTA 3216 Thr Glu Ile Gln Thr Leu Tyr Asn Asn Glu Pro Asn Ala Asn Ile Leu 1060 1065 1070 AAG GAT TTT TGG GGA AAT TAT TTG CTT TAT GAC AAA GAA TAC TAT TTA 3264 Lys Asp Phe Trp Gly Asn Tyr Leu Leu Tyr Asp Lys Glu Tyr Tyr Leu 1075 1080 1085 TTA AAT GTG TTA AAA CCA AAT AAC TTT ATT AAT AGG AGA ACA GAT TCT 3312 Leu Asn Val Leu Lys Pro Asn Asn Phe Ile Asn Arg Arg Thr Asp Ser 1090 1095 1100 ACT TTA AGC ATT AAT AAT ATA AGA AGC ACT ATT CTT TTA GCT AAT AGA 3360 Thr Leu Ser Ile Asn Asn Ile Arg Ser Thr Ile Leu Leu Ala Asn Arg 1105 1110 1115 1120 TTA TAT AGT GGA ATA AAA GTT AAA ATA CAA AGA GTT AAT AAT AGT AGT 3408 Leu Tyr Ser Gly Ile Lys Val Lys Ile Gln Arg Val Asn Asn Ser Ser 1125 1130 1135 ACT AAC GAT AAT CTT GTT AGA AAG AAT GAT CAG GTA TAT ATT AAT TTT 3456 Thr Asn Asp Asn Leu Val Arg Lys Asn Asp Gln Val Tyr Ile Asn Phe 1140 1145 1150 GTA GCC AGC AAA ACT CAC TTA CTT CCA TTA TAT GCT GAT ACA GCT ACC 3504 Val Ala Ser Lys Thr His Leu Leu Pro Leu Tyr Ala Asp Thr Ala Thr 1155 1160 1165 ACA AAT AAA GAG AAA ACA ATA AAA ATA TCA TCA TCT GGC AAT AGA TTT 3552 Thr Asn Lys Glu Lys Thr Ile Lys Ile Ser Ser Ser Gly Asn Arg Phe 1170 1175 1180 AAT CAA GTA GTA GTT ATG AAT TCA GTA GGA TGT ACA ATG AAT TTT AAA 3600 Asn Gln Val Val Val Met Asn Ser Val Gly Cys Thr Met Asn Phe Lys 1185 1190 1195 1200 AAT AAT AAT GGA AAT AAT ATT GGG TTG TTA GGT TTC AAG GCA GAT ACT 3648 Asn Asn Asn Gly Asn Asn Ile Gly Leu Leu Gly Phe Lys Ala Asp Thr 1205 1210 1215 GTA GTT GCT AGT ACT TGG TAT TAT ACA CAT ATG AGA GAT AAT ACA AAC 3696 Val Val Ala Ser Thr Trp Tyr Tyr Thr His Met Arg Asp Asn Thr Asn 1220 1225 1230 AGC AAT GGA TTT TTT TGG AAC TTT ATT TCT GAA GAA CAT GGA TGG CAA 3744 Ser Asn Gly Phe Phe Trp Asn Phe Ile Ser Glu Glu His Gly Trp Gln 1235 1240 1245 GAA AAA TAA 3753 Glu Lys 1250

50 Met Pro Thr Ile Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asn Arg 1 5 10 15 Thr Ile Leu Tyr Ile Lys Pro Gly Gly Cys Gln Gln Phe Tyr Lys Ser 20 25 30 Phe Asn Ile Met Lys Asn Ile Trp Ile Ile Pro Glu Arg Asn Val Ile 35 40 45 Gly Thr Ile Pro Gln Asp Phe Leu Pro Pro Thr Ser Leu Lys Asn Gly 50 55 60 Asp Ser Ser Tyr Tyr Asp Pro Asn Tyr Leu Gln Ser Asp Gln Glu Lys 65 70 75 80 Asp Lys Phe Leu Lys Ile Val Thr Lys Ile Phe Asn Arg Ile Asn Asp 85 90 95 Asn Leu Ser Gly Arg Ile Leu Leu Glu Glu Leu Ser Lys Ala Asn Pro 100 105 110 Tyr Leu Gly Asn Asp Asn Thr Pro Asp Gly Asp Phe Ile Ile Asn Asp 115 120 125 Ala Ser Ala Val Pro Ile Gln Phe Ser Asn Gly Ser Gln Ser Ile Leu 130 135 140 Leu Pro Asn Val Ile Ile Met Gly Ala Glu Pro Asp Leu Phe Glu Thr 145 150 155 160 Asn Ser Ser Asn Ile Ser Leu Arg Asn Asn Tyr Met Pro Ser Asn His 165 170 175 Gly Phe Gly Ser Ile Ala Ile Val Thr Phe Ser Pro Glu Tyr Ser Phe 180 185 190 Arg Phe Lys Asp Asn Ser Met Asn Glu Phe Ile Gln Asp Pro Ala Leu 195 200 205 Thr Leu Met His Glu Leu Ile His Ser Leu His Gly Leu Tyr Gly Ala 210 215 220 Lys Gly Ile Thr Thr Lys Tyr Thr Ile Thr Gln Lys Gln Asn Pro Leu 225 230 235 240 Ile Thr Asn Ile Arg Gly Thr Asn Ile Glu Glu Phe Leu Thr Phe Gly 245 250 255 Gly Thr Asp Leu Asn Ile Ile Thr Ser Ala Gln Ser Asn Asp Ile Tyr 260 265 270 Thr Asn Leu Leu Ala Asp Tyr Lys Lys Ile Ala Ser Lys Leu Ser Lys 275 280 285 Val Gln Val Ser Asn Pro Leu Leu Asn Pro Tyr Lys Asp Val Phe Glu 290 295 300 Ala Lys Tyr Gly Leu Asp Lys Asp Ala Ser Gly Ile Tyr Ser Val Asn 305 310 315 320 Ile Asn Lys Phe Asn Asp Ile Phe Lys Lys Leu Tyr Ser Phe Thr Glu 325 330 335 Phe Asp Leu Ala Thr Lys Phe Gln Val Lys Cys Arg Gln Thr Tyr Ile 340 345 350 Gly Gln Tyr Lys Tyr Phe Lys Leu Ser Asn Leu Leu Asn Asp Ser Ile 355 360 365 Tyr Asn Ile Ser Glu Gly Tyr Asn Ile Asn Asn Leu Lys Val Asn Phe 370 375 380 Arg Gly Gln Asn Ala Asn Leu Asn Pro Arg Ile Ile Thr Pro Ile Thr 385 390 395 400 Gly Arg Gly Leu Val Lys Lys Ile Ile Arg Phe Cys Lys Asn Ile Val 405 410 415 Ser Val Lys Gly Ile Arg Lys Ser Ile Cys Ile Glu Ile Asn Asn Gly 420 425 430 Glu Leu Phe Phe Val Ala Ser Glu Asn Ser Tyr Asn Asp Asp Asn Ile 435 440 445 Asn Thr Pro Lys Glu Ile Asp Asp Thr Val Thr Ser Asn Asn Asn Tyr 450 455 460 Glu Asn Asp Leu Asp Gln Val Ile Leu Asn Phe Asn Ser Glu Ser Ala 465 470 475 480 Pro Gly Leu Ser Asp Glu Lys Leu Asn Leu Thr Ile Gln Asn Asp Ala 485 490 495 Tyr Ile Pro Lys Tyr Asp Ser Asn Gly Thr Ser Asp Ile Glu Gln His 500 505 510 Asp Val Asn Glu Leu Asn Val Phe Phe Tyr Leu Asp Ala Gln Lys Val 515 520 525 Pro Glu Gly Glu Asn Asn Val Asn Leu Thr Ser Ser Ile Asp Thr Ala 530 535 540 Leu Leu Glu Gln Pro Lys Ile Tyr Thr Phe Phe Ser Ser Glu Phe Ile 545 550 555 560 Asn Asn Val Asn Lys Pro Val Gln Ala Ala Leu Phe Val Ser Trp Ile 565 570 575 Gln Gln Val Leu Val Asp Phe Thr Thr Glu Ala Asn Gln Lys Ser Thr 580 585 590 Val Asp Lys Ile Ala Asp Ile Ser Ile Val Val Pro Tyr Ile Gly Leu 595 600 605 Ala Leu Asn Ile Gly Asn Glu Ala Gln Lys Gly Asn Phe Lys Asp Ala 610 615 620 Leu Glu Leu Leu Gly Ala Gly Ile Leu Leu Glu Phe Glu Pro Glu Leu 625 630 635 640 Leu Ile Pro Thr Ile Leu Val Phe Thr Ile Lys Ser Phe Leu Gly Ser 645 650 655 Ser Asp Asn Lys Asn Lys Val Ile Lys Ala Ile Asn Asn Ala Leu Lys 660 665 670 Glu Arg Asp Glu Lys Trp Lys Glu Val Tyr Ser Phe Ile Val Ser Asn 675 680 685 Trp Met Thr Lys Ile Asn Thr Gln Phe Asn Lys Arg Lys Glu Gln Met 690 695 700 Tyr Gln Ala Leu Gln Asn Gln Val Asn Ala Leu Lys Ala Ile Ile Glu 705 710 715 720 Ser Lys Tyr Asn Ser Tyr Thr Leu Glu Glu Lys Asn Glu Leu Thr Asn 725 730 735 Lys Tyr Asp Ile Glu Gln Ile Glu Asn Glu Leu Asn Gln Lys Val Ser 740 745 750 Ile Ala Met Asn Asn Ile Asp Arg Phe Leu Thr Glu Ser Ser Ile Ser 755 760 765 Tyr Leu Met Lys Leu Ile Asn Glu Val Lys Ile Asn Lys Leu Arg Glu 770 775 780 Tyr Asp Glu Asn Val Lys Thr Tyr Leu Leu Asp Tyr Ile Ile Lys His 785 790 795 800 Gly Ser Ile Leu Gly Glu Ser Gln Gln Glu Leu Asn Ser Met Val Ile 805 810 815 Asp Thr Leu Asn Asn Ser Ile Pro Phe Lys Leu Ser Ser Tyr Thr Asp 820 825 830 Asp Lys Ile Leu Ile Ser Tyr Phe Asn Lys Phe Phe Lys Arg Ile Lys 835 840 845 Ser Ser Ser Val Leu Asn Met Arg Tyr Lys Asn Asp Lys Tyr Val Asp 850 855 860 Thr Ser Gly Tyr Asp Ser Asn Ile Asn Ile Asn Gly Asp Val Tyr Lys 865 870 875 880 Tyr Pro Thr Asn Lys Asn Gln Phe Gly Ile Tyr Asn Asp Lys Leu Ser 885 890 895 Glu Val Asn Ile Ser Gln Asn Asp Tyr Ile Ile Tyr Asp Asn Lys Tyr 900 905 910 Lys Asn Phe Ser Ile Ser Phe Trp Val Arg Ile Pro Asn Tyr Asp Asn 915 920 925 Lys Ile Val Asn Val Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Arg 930 935 940 Asp Asn Asn Ser Gly Trp Lys Val Ser Leu Asn His Asn Glu Ile Ile 945 950 955 960 Trp Thr Leu Gln Asp Asn Ser Gly Ile Asn Gln Lys Leu Ala Phe Asn 965 970 975 Tyr Gly Asn Ala Asn Gly Ile Ser Asp Tyr Ile Asn Lys Trp Ile Phe 980 985 990 Val Thr Ile Thr Asn Asp Arg Leu Gly Asp Ser Lys Leu Tyr Ile Asn 995 1000 1005 Gly Asn Leu Ile Asp Lys Lys Ser Ile Leu Asn Leu Gly Asn Ile His 1010 1015 1020 Val Ser Asp Asn Ile Leu Phe Lys Ile Val Asn Cys Ser Tyr Thr Arg 1025 1030 1035 1040 Tyr Ile Gly Ile Arg Tyr Phe Asn Ile Phe Asp Lys Glu Leu Asp Glu 1045 1050 1055 Thr Glu Ile Gln Thr Leu Tyr Asn Asn Glu Pro Asn Ala Asn Ile Leu 1060 1065 1070 Lys Asp Phe Trp Gly Asn Tyr Leu Leu Tyr Asp Lys Glu Tyr Tyr Leu 1075 1080 1085 Leu Asn Val Leu Lys Pro Asn Asn Phe Ile Asn Arg Arg Thr Asp Ser 1090 1095 1100 Thr Leu Ser Ile Asn Asn Ile Arg Ser Thr Ile Leu Leu Ala Asn Arg 1105 1110 1115 1120 Leu Tyr Ser Gly Ile Lys Val Lys Ile Gln Arg Val Asn Asn Ser Ser 1125 1130 1135 Thr Asn Asp Asn Leu Val Arg Lys Asn Asp Gln Val Tyr Ile Asn Phe 1140 1145 1150 Val Ala Ser Lys Thr His Leu Leu Pro Leu Tyr Ala Asp Thr Ala Thr 1155 1160 1165 Thr Asn Lys Glu Lys Thr Ile Lys Ile Ser Ser Ser Gly Asn Arg Phe 1170 1175 1180 Asn Gln Val Val Val Met Asn Ser Val Gly Cys Thr Met Asn Phe Lys 1185 1190 1195 1200 Asn Asn Asn Gly Asn Asn Ile Gly Leu Leu Gly Phe Lys Ala Asp Thr 1205 1210 1215 Val Val Ala Ser Thr Trp Tyr Tyr Thr His Met Arg Asp Asn Thr Asn 1220 1225 1230 Ser Asn Gly Phe Phe Trp Asn Phe Ile Ser Glu Glu His Gly Trp Gln 1235 1240 1245 Glu Lys 1250

51 ATG CCA AAA ATT AAT AGT TTT AAT TAT AAT GAT CCT GTT AAT GAT AGA 48 Met Pro Lys Ile Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asp Arg 1 5 10 15 ACA ATT TTA TAT ATT AAA CCA GGC GGT TGT CAA GAA TTT TAT AAA TCA 96 Thr Ile Leu Tyr Ile Lys Pro Gly Gly Cys Gln Glu Phe Tyr Lys Ser 20 25 30 TTT AAT ATT ATG AAA AAT ATT TGG ATA ATT CCA GAG AGA AAT GTA ATT 144 Phe Asn Ile Met Lys Asn Ile Trp Ile Ile Pro Glu Arg Asn Val Ile 35 40 45 GGT ACA ACC CCC CAA GAT TTT CAT CCG CCT ACT TCA TTA AAA AAT GGA 192 Gly Thr Thr Pro Gln Asp Phe His Pro Pro Thr Ser Leu Lys Asn Gly 50 55 60 GAT AGT AGT TAT TAT GAC CCT AAT TAT TTA CAA AGT GAT GAA GAA AAG 240 Asp Ser Ser Tyr Tyr Asp Pro Asn Tyr Leu Gln Ser Asp Glu Glu Lys 65 70 75 80 GAT AGA TTT TTA AAA ATA GTC ACA AAA ATA TTT AAT AGA ATA AAT AAT 288 Asp Arg Phe Leu Lys Ile Val Thr Lys Ile Phe Asn Arg Ile Asn Asn 85 90 95 AAT CTT TCA GGA GGG ATT TTA TTA GAA GAA CTG TCA AAA GCT AAT CCA 336 Asn Leu Ser Gly Gly Ile Leu Leu Glu Glu Leu Ser Lys Ala Asn Pro 100 105 110 TAT TTA GGG AAT GAT AAT ACT CCA GAT AAT CAA TTC CAT ATT GGT GAT 384 Tyr Leu Gly Asn Asp Asn Thr Pro Asp Asn Gln Phe His Ile Gly Asp 115 120 125 GCA TCA GCA GTT GAG ATT AAA TTC TCA AAT GGT AGC CAA GAC ATA CTA 432 Ala Ser Ala Val Glu Ile Lys Phe Ser Asn Gly Ser Gln Asp Ile Leu 130 135 140 TTA CCT AAT GTT ATT ATA ATG GGA GCA GAG CCT GAT TTA TTT GAA ACT 480 Leu Pro Asn Val Ile Ile Met Gly Ala Glu Pro Asp Leu Phe Glu Thr 145 150 155 160 AAC AGT TCC AAT ATT TCT CTA AGA AAT AAT TAT ATG CCA AGC AAT CAC 528 Asn Ser Ser Asn Ile Ser Leu Arg Asn Asn Tyr Met Pro Ser Asn His 165 170 175 GGT TTT GGA TCA ATA GCT ATA GTA ACA TTC TCA CCT GAA TAT TCT TTT 576 Gly Phe Gly Ser Ile Ala Ile Val Thr Phe Ser Pro Glu Tyr Ser Phe 180 185 190 AGA TTT AAT GAT AAT AGT ATG AAT GAA TTT ATT CAA GAT CCT GCT CTT 624 Arg Phe Asn Asp Asn Ser Met Asn Glu Phe Ile Gln Asp Pro Ala Leu 195 200 205 ACA TTA ATG CAT GAA TTA ATA CAT TCA TTA CAT GGA CTA TAT GGG GCT 672 Thr Leu Met His Glu Leu Ile His Ser Leu His Gly Leu Tyr Gly Ala 210 215 220 AAA GGG ATT ACT ACA AAG TAT ACT ATA ACA CAA AAA CAA AAT CCC CTA 720 Lys Gly Ile Thr Thr Lys Tyr Thr Ile Thr Gln Lys Gln Asn Pro Leu 225 230 235 240 ATA ACA AAT ATA AGA GGT ACA AAT ATT GAA GAA TTC TTA ACT TTT GGA 768 Ile Thr Asn Ile Arg Gly Thr Asn Ile Glu Glu Phe Leu Thr Phe Gly 245 250 255 GGT ACT GAT TTA AAC ATT ATT ACT AGT GCT CAG TCC AAT GAT ATC TAT 816 Gly Thr Asp Leu Asn Ile Ile Thr Ser Ala Gln Ser Asn Asp Ile Tyr 260 265 270 ACT AAT CTT CTA GCT GAT TAT AAA AAA ATA GCG TCT AAA CTT AGC AAA 864 Thr Asn Leu Leu Ala Asp Tyr Lys Lys Ile Ala Ser Lys Leu Ser Lys 275 280 285 GTA CAA GTA TCT AAT CCA CTA CTT AAT CCT TAT AAA GAT GTT TTT GAA 912 Val Gln Val Ser Asn Pro Leu Leu Asn Pro Tyr Lys Asp Val Phe Glu 290 295 300 GCA AAG TAT GGA TTA GAT AAA GAT GCT AGC GGA ATT TAT TCG GTA AAT 960 Ala Lys Tyr Gly Leu Asp Lys Asp Ala Ser Gly Ile Tyr Ser Val Asn 305 310 315 320 ATA AAC AAA TTT AAT GAT ATT TTT AAA AAA TTA TAC AGC TTT ACG GAA 1008 Ile Asn Lys Phe Asn Asp Ile Phe Lys Lys Leu Tyr Ser Phe Thr Glu 325 330 335 TTT GAT TTA GCA ACT AAA TTT CAA GTT AAA TGT AGG CAA ACT TAT ATT 1056 Phe Asp Leu Ala Thr Lys Phe Gln Val Lys Cys Arg Gln Thr Tyr Ile 340 345 350 GGA CAG TAT AAA TAC TTC AAA CTT TCA AAC TTG TTA AAT GAT TCT ATT 1104 Gly Gln Tyr Lys Tyr Phe Lys Leu Ser Asn Leu Leu Asn Asp Ser Ile 355 360 365 TAT AAT ATA TCA GAA GGC TAT AAT ATA AAT AAT TTA AAG GTA AAT TTT 1152 Tyr Asn Ile Ser Glu Gly Tyr Asn Ile Asn Asn Leu Lys Val Asn Phe 370 375 380 AGA GGA CAG AAT GCA AAT TTA AAT CCT AGA ATT ATT ACA CCA ATT ACA 1200 Arg Gly Gln Asn Ala Asn Leu Asn Pro Arg Ile Ile Thr Pro Ile Thr 385 390 395 400 GGT AGA GGA CTA GTA AAA AAA ATC ATT AGA TTT TGT AAA AAT ATT GTT 1248 Gly Arg Gly Leu Val Lys Lys Ile Ile Arg Phe Cys Lys Asn Ile Val 405 410 415 TCT GTA AAA GGC ATA AGG AAA TCA ATA TGT ATC GAA ATA AAT AAT GGT 1296 Ser Val Lys Gly Ile Arg Lys Ser Ile Cys Ile Glu Ile Asn Asn Gly 420 425 430 GAG TTA TTT TTT GTG GCT TCC GAG AAT AGT TAT AAT GAT GAT AAT ATA 1344 Glu Leu Phe Phe Val Ala Ser Glu Asn Ser Tyr Asn Asp Asp Asn Ile 435 440 445 AAT ACT CCT AAA GAA ATT GAC GAT ACA GTA ACT TCA AAT AAT AAT TAT 1392 Asn Thr Pro Lys Glu Ile Asp Asp Thr Val Thr Ser Asn Asn Asn Tyr 450 455 460 GAA AAT GAT TTA GAT CAG GTT ATT TTA AAT TTT AAT AGT GAA TCA GCA 1440 Glu Asn Asp Leu Asp Gln Val Ile Leu Asn Phe Asn Ser Glu Ser Ala 465 470 475 480 CCT GGA CTT TCA GAT GAA AAA TTA AAT TTA ACT ATC CAA AAT GAT GCT 1488 Pro Gly Leu Ser Asp Glu Lys Leu Asn Leu Thr Ile Gln Asn Asp Ala 485 490 495 TAT ATA CCA AAA TAT GAT TCT AAT GGA ACA AGT GAT ATA GAA CAA CAT 1536 Tyr Ile Pro Lys Tyr Asp Ser Asn Gly Thr Ser Asp Ile Glu Gln His 500 505 510 GAT GTT AAT GAA CTT AAT GTA TTT TTC TAT TTA GAT GCA CAG AAA GTG 1584 Asp Val Asn Glu Leu Asn Val Phe Phe Tyr Leu Asp Ala Gln Lys Val 515 520 525 CCC GAA GGT GAA AAT AAT GTC AAT CTC ACC TCT TCA ATT GAT ACA GCA 1632 Pro Glu Gly Glu Asn Asn Val Asn Leu Thr Ser Ser Ile Asp Thr Ala 530 535 540 TTA TTA GAA CAA CCT AAA ATA TAT ACA TTT TTT TCA TCA GAA TTT ATT 1680 Leu Leu Glu Gln Pro Lys Ile Tyr Thr Phe Phe Ser Ser Glu Phe Ile 545 550 555 560 AAT AAT GTC AAT AAA CCT GTG CAA GCA GCA TTA TTT GTA AGC TGG ATA 1728 Asn Asn Val Asn Lys Pro Val Gln Ala Ala Leu Phe Val Ser Trp Ile 565 570 575 CAA CAA GTG TTA GTA GAT TTT ACT ACT GAA GCT AAC CAA AAA AGT ACT 1776 Gln Gln Val Leu Val Asp Phe Thr Thr Glu Ala Asn Gln Lys Ser Thr 580 585 590 GTT GAT AAA ATT GCA GAT ATT TCT ATA GTT GTT CCA TAT ATA GGT CTT 1824 Val Asp Lys Ile Ala Asp Ile Ser Ile Val Val Pro Tyr Ile Gly Leu 595 600 605 GCT TTA AAT ATA GGA AAT GAA GCA CAA AAA GGA AAT TTT AAA GAT GCA 1872 Ala Leu Asn Ile Gly Asn Glu Ala Gln Lys Gly Asn Phe Lys Asp Ala 610 615 620 CTT GAA TTA TTA GGA GCA GGT ATT TTA TTA GAA TTT GAA CCC GAG CTT 1920 Leu Glu Leu Leu Gly Ala Gly Ile Leu Leu Glu Phe Glu Pro Glu Leu 625 630 635 640 TTA ATT CCT ACA ATT TTA GTA TTC ACG ATA AAA TCT TTT TTA GGT TCA 1968 Leu Ile Pro Thr Ile Leu Val Phe Thr Ile Lys Ser Phe Leu Gly Ser 645 650 655 TCT GAT AAT AAA AAT AAA GTT ATT AAA GCA ATA AAT AAT GCA TTG AAA 2016 Ser Asp Asn Lys Asn Lys Val Ile Lys Ala Ile Asn Asn Ala Leu Lys 660 665 670 GAA AGA GAT GAA AAA TGG AAA GAA GTA TAT AGT TTT ATA GTA TCG AAT 2064 Glu Arg Asp Glu Lys Trp Lys Glu Val Tyr Ser Phe Ile Val Ser Asn 675 680 685 TGG ATG ACT AAA ATT AAT ACA CAA TTT AAT AAA AGA AAA GAA CAA ATG 2112 Trp Met Thr Lys Ile Asn Thr Gln Phe Asn Lys Arg Lys Glu Gln Met 690 695 700 TAT CAA GCT TTA CAA AAT CAA GTA AAT GCA ATT AAA ACA ATA ATA GAA 2160 Tyr Gln Ala Leu Gln Asn Gln Val Asn Ala Ile Lys Thr Ile Ile Glu 705 710 715 720 TCT AAG TAT AAT AGT TAT ACT TTA GAG GAA AAA AAT GAG CTT ACA AAT 2208 Ser Lys Tyr Asn Ser Tyr Thr Leu Glu Glu Lys Asn Glu Leu Thr Asn 725 730 735 AAA TAT GAT ATT AAG CAA ATA GAA AAT GAA CTT AAT CAA AAG GTT TCT 2256 Lys Tyr Asp Ile Lys Gln Ile Glu Asn Glu Leu Asn Gln Lys Val Ser 740 745 750 ATA GCA ATG AAT AAT ATA GAC AGG TTC TTA ACT GAA AGT TCT ATA TCC 2304 Ile Ala Met Asn Asn Ile Asp Arg Phe Leu Thr Glu Ser Ser Ile Ser 755 760 765 TAT TTA ATG AAA TTA ATA AAT GAA GTA AAA ATT AAT AAA TTA AGA GAA 2352 Tyr Leu Met Lys Leu Ile Asn Glu Val Lys Ile Asn Lys Leu Arg Glu 770 775 780 TAT GAT GAG AAT GTC AAA ACG TAT TTA TTG AAT TAT ATT ATA CAA CAT 2400 Tyr Asp Glu Asn Val Lys Thr Tyr Leu Leu Asn Tyr Ile Ile Gln His 785 790 795 800 GGA TCA ATC TTG GGA GAG AGT CAG CAA GAA CTA AAT TCT ATG GTA ACT 2448 Gly Ser Ile Leu Gly Glu Ser Gln Gln Glu Leu Asn Ser Met Val Thr 805 810 815 GAT ACC CTA AAT AAT AGT ATT CCT TTT AAG CTT TCT TCT TAT ACA GAT 2496 Asp Thr Leu Asn Asn Ser Ile Pro Phe Lys Leu Ser Ser Tyr Thr Asp 820 825 830 GAT AAA ATT TTA ATT TCA TAT TTT AAT AAA TTC TTT AAG AGA ATT AAA 2544 Asp Lys Ile Leu Ile Ser Tyr Phe Asn Lys Phe Phe Lys Arg Ile Lys 835 840 845 AGT AGT TCA GTT TTA AAT ATG AGA TAT AAA AAT GAT AAA TAC GTA GAT 2592 Ser Ser Ser Val Leu Asn Met Arg Tyr Lys Asn Asp Lys Tyr Val Asp 850 855 860 ACT TCA GGA TAT GAT TCA AAT ATA AAT ATT AAT GGA GAT GTA TAT AAA 2640 Thr Ser Gly Tyr Asp Ser Asn Ile Asn Ile Asn Gly Asp Val Tyr Lys 865 870 875 880 TAT CCA ACT AAT AAA AAT CAA TTT GGA ATA TAT AAT GAT AAA CTT AGT 2688 Tyr Pro Thr Asn Lys Asn Gln Phe Gly Ile Tyr Asn Asp Lys Leu Ser 885 890 895 GAA GTT AAT ATA TCT CAA AAT GAT TAC ATT ATA TAT GAT AAT AAA TAT 2736 Glu Val Asn Ile Ser Gln Asn Asp Tyr Ile Ile Tyr Asp Asn Lys Tyr 900 905 910 AAA AAT TTT AGT ATT AGT TTT TGG GTA AGA ATT CCT AAC TAT GAT AAT 2784 Lys Asn Phe Ser Ile Ser Phe Trp Val Arg Ile Pro Asn Tyr Asp Asn 915 920 925 AAG ATA GTA AAT GTT AAT AAT GAA TAC ACT ATA ATA AAT TGT ATG AGA 2832 Lys Ile Val Asn Val Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Arg 930 935 940 GAT AAT AAT TCA GGA TGG AAA GTA TCT CTT AAT CAT AAT GAA ATA ATT 2880 Asp Asn Asn Ser Gly Trp Lys Val Ser Leu Asn His Asn Glu Ile Ile 945 950 955 960 TGG ACA TTG CAA GAT AAT GCA GGA ATT AAT CAA AAA TTA GCA TTT AAC 2928 Trp Thr Leu Gln Asp Asn Ala Gly Ile Asn Gln Lys Leu Ala Phe Asn 965 970 975 TAT GGT AAC GCA AAT GGT ATT TCT GAT TAT ATA AAT AAG TGG ATT TTT 2976 Tyr Gly Asn Ala Asn Gly Ile Ser Asp Tyr Ile Asn Lys Trp Ile Phe 980 985 990 GTA ACT ATA ACT AAT GAT AGA TTA GGA GAT TCT AAA CTT TAT ATT AAT 3024 Val Thr Ile Thr Asn Asp Arg Leu Gly Asp Ser Lys Leu Tyr Ile Asn 995 1000 1005 GGA AAT TTA ATA GAT CAA AAA TCA ATT TTA AAT TTA GGT AAT ATT CAT 3072 Gly Asn Leu Ile Asp Gln Lys Ser Ile Leu Asn Leu Gly Asn Ile His 1010 1015 1020 GTT AGT GAC AAT ATA TTA TTT AAA ATA GTT AAT TGT AGT TAT ACA AGA 3120 Val Ser Asp Asn Ile Leu Phe Lys Ile Val Asn Cys Ser Tyr Thr Arg 1025 1030 1035 1040 TAT ATT GGT ATT AGA TAT TTT AAT ATT TTT GAT AAA GAA TTA GAT GAA 3168 Tyr Ile Gly Ile Arg Tyr Phe Asn Ile Phe Asp Lys Glu Leu Asp Glu 1045 1050 1055 ACA GAA ATT CAA ACT TTA TAT AGC AAT GAA CCT AAT ACA AAT ATT TTG 3216 Thr Glu Ile Gln Thr Leu Tyr Ser Asn Glu Pro Asn Thr Asn Ile Leu 1060 1065 1070 AAG GAT TTT TGG GGA AAT TAT TTG CTT TAT GAC AAA GAA TAC TAT TTA 3264 Lys Asp Phe Trp Gly Asn Tyr Leu Leu Tyr Asp Lys Glu Tyr Tyr Leu 1075 1080 1085 TTA AAT GTG TTA AAA CCA AAT AAC TTT ATT GAT AGG AGA AAA GAT TCT 3312 Leu Asn Val Leu Lys Pro Asn Asn Phe Ile Asp Arg Arg Lys Asp Ser 1090 1095 1100 ACT TTA AGC ATT AAT AAT ATA AGA AGC ACT ATT CTT TTA GCT AAT AGA 3360 Thr Leu Ser Ile Asn Asn Ile Arg Ser Thr Ile Leu Leu Ala Asn Arg 1105 1110 1115 1120 TTA TAT AGT GGA ATA AAA GTT AAA ATA CAA AGA GTT AAT AAT AGT AGT 3408 Leu Tyr Ser Gly Ile Lys Val Lys Ile Gln Arg Val Asn Asn Ser Ser 1125 1130 1135 ACT AAC GAT AAT CTT GTT AGA AAG AAT GAT CAG GTA TAT ATT AAT TTT 3456 Thr Asn Asp Asn Leu Val Arg Lys Asn Asp Gln Val Tyr Ile Asn Phe 1140 1145 1150 GTA GCC AGC AAA ACT CAC TTA TTT CCA TTA TAT GCT GAT ACA GCT ACC 3504 Val Ala Ser Lys Thr His Leu Phe Pro Leu Tyr Ala Asp Thr Ala Thr 1155 1160 1165 ACA AAT AAA GAG AAA ACA ATA AAA ATA TCA TCA TCT GGC AAT AGA TTT 3552 Thr Asn Lys Glu Lys Thr Ile Lys Ile Ser Ser Ser Gly Asn Arg Phe 1170 1175 1180 AAT CAA GTA GTA GTT ATG AAT TCA GTA GGA AAT AAT TGT ACA ATG AAT 3600 Asn Gln Val Val Val Met Asn Ser Val Gly Asn Asn Cys Thr Met Asn 1185 1190 1195 1200 TTT AAA AAT AAT AAT GGA AAT AAT ATT GGG TTG TTA GGT TTC AAG GCA 3648 Phe Lys Asn Asn Asn Gly Asn Asn Ile Gly Leu Leu Gly Phe Lys Ala 1205 1210 1215 GAT ACT GTA GTT GCT AGT ACT TGG TAT TAT ACA CAT ATG AGA GAT CAT 3696 Asp Thr Val Val Ala Ser Thr Trp Tyr Tyr Thr His Met Arg Asp His 1220 1225 1230 ACA AAC AGC AAT GGA TGT TTT TGG AAC TTT ATT TCT GAA GAA CAT GGA 3744 Thr Asn Ser Asn Gly Cys Phe Trp Asn Phe Ile Ser Glu Glu His Gly 1235 1240 1245 TGG CAA GAA AAA TAA 3759 Trp Gln Glu Lys 1250

52 Met Pro Lys Ile Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asp Arg 1 5 10 15 Thr Ile Leu Tyr Ile Lys Pro Gly Gly Cys Gln Glu Phe Tyr Lys Ser 20 25 30 Phe Asn Ile Met Lys Asn Ile Trp Ile Ile Pro Glu Arg Asn Val Ile 35 40 45 Gly Thr Thr Pro Gln Asp Phe His Pro Pro Thr Ser Leu Lys Asn Gly 50 55 60 Asp Ser Ser Tyr Tyr Asp Pro Asn Tyr Leu Gln Ser Asp Glu Glu Lys 65 70 75 80 Asp Arg Phe Leu Lys Ile Val Thr Lys Ile Phe Asn Arg Ile Asn Asn 85 90 95 Asn Leu Ser Gly Gly Ile Leu Leu Glu Glu Leu Ser Lys Ala Asn Pro 100 105 110 Tyr Leu Gly Asn Asp Asn Thr Pro Asp Asn Gln Phe His Ile Gly Asp 115 120 125 Ala Ser Ala Val Glu Ile Lys Phe Ser Asn Gly Ser Gln Asp Ile Leu 130 135 140 Leu Pro Asn Val Ile Ile Met Gly Ala Glu Pro Asp Leu Phe Glu Thr 145 150 155 160 Asn Ser Ser Asn Ile Ser Leu Arg Asn Asn Tyr Met Pro Ser Asn His 165 170 175 Gly Phe Gly Ser Ile Ala Ile Val Thr Phe Ser Pro Glu Tyr Ser Phe 180 185 190 Arg Phe Asn Asp Asn Ser Met Asn Glu Phe Ile Gln Asp Pro Ala Leu 195 200 205 Thr Leu Met His Glu Leu Ile His Ser Leu His Gly Leu Tyr Gly Ala 210 215 220 Lys Gly Ile Thr Thr Lys Tyr Thr Ile Thr Gln Lys Gln Asn Pro Leu 225 230 235 240 Ile Thr Asn Ile Arg Gly Thr Asn Ile Glu Glu Phe Leu Thr Phe Gly 245 250 255 Gly Thr Asp Leu Asn Ile Ile Thr Ser Ala Gln Ser Asn Asp Ile Tyr 260 265 270 Thr Asn Leu Leu Ala Asp Tyr Lys Lys Ile Ala Ser Lys Leu Ser Lys 275 280 285 Val Gln Val Ser Asn Pro Leu Leu Asn Pro Tyr Lys Asp Val Phe Glu 290 295 300 Ala Lys Tyr Gly Leu Asp Lys Asp Ala Ser Gly Ile Tyr Ser Val Asn 305 310 315 320 Ile Asn Lys Phe Asn Asp Ile Phe Lys Lys Leu Tyr Ser Phe Thr Glu 325 330 335 Phe Asp Leu Ala Thr Lys Phe Gln Val Lys Cys Arg Gln Thr Tyr Ile 340 345 350 Gly Gln Tyr Lys Tyr Phe Lys Leu Ser Asn Leu Leu Asn Asp Ser Ile 355 360 365 Tyr Asn Ile Ser Glu Gly Tyr Asn Ile Asn Asn Leu Lys Val Asn Phe 370 375 380 Arg Gly Gln Asn Ala Asn Leu Asn Pro Arg Ile Ile Thr Pro Ile Thr 385 390 395 400 Gly Arg Gly Leu Val Lys Lys Ile Ile Arg Phe Cys Lys Asn Ile Val 405 410 415 Ser Val Lys Gly Ile Arg Lys Ser Ile Cys Ile Glu Ile Asn Asn Gly 420 425 430 Glu Leu Phe Phe Val Ala Ser Glu Asn Ser Tyr Asn Asp Asp Asn Ile 435 440 445 Asn Thr Pro Lys Glu Ile Asp Asp Thr Val Thr Ser Asn Asn Asn Tyr 450 455 460 Glu Asn Asp Leu Asp Gln Val Ile Leu Asn Phe Asn Ser Glu Ser Ala 465 470 475 480 Pro Gly Leu Ser Asp Glu Lys Leu Asn Leu Thr Ile Gln Asn Asp Ala 485 490 495 Tyr Ile Pro Lys Tyr Asp Ser Asn Gly Thr Ser Asp Ile Glu Gln His 500 505 510 Asp Val Asn Glu Leu Asn Val Phe Phe Tyr Leu Asp Ala Gln Lys Val 515 520 525 Pro Glu Gly Glu Asn Asn Val Asn Leu Thr Ser Ser Ile Asp Thr Ala 530 535 540 Leu Leu Glu Gln Pro Lys Ile Tyr Thr Phe Phe Ser Ser Glu Phe Ile 545 550 555 560 Asn Asn Val Asn Lys Pro Val Gln Ala Ala Leu Phe Val Ser Trp Ile 565 570 575 Gln Gln Val Leu Val Asp Phe Thr Thr Glu Ala Asn Gln Lys Ser Thr 580 585 590 Val Asp Lys Ile Ala Asp Ile Ser Ile Val Val Pro Tyr Ile Gly Leu 595 600 605 Ala Leu Asn Ile Gly Asn Glu Ala Gln Lys Gly Asn Phe Lys Asp Ala 610 615 620 Leu Glu Leu Leu Gly Ala Gly Ile Leu Leu Glu Phe Glu Pro Glu Leu 625 630 635 640 Leu Ile Pro Thr Ile Leu Val Phe Thr Ile Lys Ser Phe Leu Gly Ser 645 650 655 Ser Asp Asn Lys Asn Lys Val Ile Lys Ala Ile Asn Asn Ala Leu Lys 660 665 670 Glu Arg Asp Glu Lys Trp Lys Glu Val Tyr Ser Phe Ile Val Ser Asn 675 680 685 Trp Met Thr Lys Ile Asn Thr Gln Phe Asn Lys Arg Lys Glu Gln Met 690 695 700 Tyr Gln Ala Leu Gln Asn Gln Val Asn Ala Ile Lys Thr Ile Ile Glu 705 710 715 720 Ser Lys Tyr Asn Ser Tyr Thr Leu Glu Glu Lys Asn Glu Leu Thr Asn 725 730 735 Lys Tyr Asp Ile Lys Gln Ile Glu Asn Glu Leu Asn Gln Lys Val Ser 740 745 750 Ile Ala Met Asn Asn Ile Asp Arg Phe Leu Thr Glu Ser Ser Ile Ser 755 760 765 Tyr Leu Met Lys Leu Ile Asn Glu Val Lys Ile Asn Lys Leu Arg Glu 770 775 780 Tyr Asp Glu Asn Val Lys Thr Tyr Leu Leu Asn Tyr Ile Ile Gln His 785 790 795 800 Gly Ser Ile Leu Gly Glu Ser Gln Gln Glu Leu Asn Ser Met Val Thr 805 810 815 Asp Thr Leu Asn Asn Ser Ile Pro Phe Lys Leu Ser Ser Tyr Thr Asp 820 825 830 Asp Lys Ile Leu Ile Ser Tyr Phe Asn Lys Phe Phe Lys Arg Ile Lys 835 840 845 Ser Ser Ser Val Leu Asn Met Arg Tyr Lys Asn Asp Lys Tyr Val Asp 850 855 860 Thr Ser Gly Tyr Asp Ser Asn Ile Asn Ile Asn Gly Asp Val Tyr Lys 865 870 875 880 Tyr Pro Thr Asn Lys Asn Gln Phe Gly Ile Tyr Asn Asp Lys Leu Ser 885 890 895 Glu Val Asn Ile Ser Gln Asn Asp Tyr Ile Ile Tyr Asp Asn Lys Tyr 900 905 910 Lys Asn Phe Ser Ile Ser Phe Trp Val Arg Ile Pro Asn Tyr Asp Asn 915 920 925 Lys Ile Val Asn Val Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Arg 930 935 940 Asp Asn Asn Ser Gly Trp Lys Val Ser Leu Asn His Asn Glu Ile Ile 945 950 955 960 Trp Thr Leu Gln Asp Asn Ala Gly Ile Asn Gln Lys Leu Ala Phe Asn 965 970 975 Tyr Gly Asn Ala Asn Gly Ile Ser Asp Tyr Ile Asn Lys Trp Ile Phe 980 985 990 Val Thr Ile Thr Asn Asp Arg Leu Gly Asp Ser Lys Leu Tyr Ile Asn 995 1000 1005 Gly Asn Leu Ile Asp Gln Lys Ser Ile Leu Asn Leu Gly Asn Ile His 1010 1015 1020 Val Ser Asp Asn Ile Leu Phe Lys Ile Val Asn Cys Ser Tyr Thr Arg 1025 1030 1035 1040 Tyr Ile Gly Ile Arg Tyr Phe Asn Ile Phe Asp Lys Glu Leu Asp Glu 1045 1050 1055 Thr Glu Ile Gln Thr Leu Tyr Ser Asn Glu Pro Asn Thr Asn Ile Leu 1060 1065 1070 Lys Asp Phe Trp Gly Asn Tyr Leu Leu Tyr Asp Lys Glu Tyr Tyr Leu 1075 1080 1085 Leu Asn Val Leu Lys Pro Asn Asn Phe Ile Asp Arg Arg Lys Asp Ser 1090 1095 1100 Thr Leu Ser Ile Asn Asn Ile Arg Ser Thr Ile Leu Leu Ala Asn Arg 1105 1110 1115 1120 Leu Tyr Ser Gly Ile Lys Val Lys Ile Gln Arg Val Asn Asn Ser Ser 1125 1130 1135 Thr Asn Asp Asn Leu Val Arg Lys Asn Asp Gln Val Tyr Ile Asn Phe 1140 1145 1150 Val Ala Ser Lys Thr His Leu Phe Pro Leu Tyr Ala Asp Thr Ala Thr 1155 1160 1165 Thr Asn Lys Glu Lys Thr Ile Lys Ile Ser Ser Ser Gly Asn Arg Phe 1170 1175 1180 Asn Gln Val Val Val Met Asn Ser Val Gly Asn Asn Cys Thr Met Asn 1185 1190 1195 1200 Phe Lys Asn Asn Asn Gly Asn Asn Ile Gly Leu Leu Gly Phe Lys Ala 1205 1210 1215 Asp Thr Val Val Ala Ser Thr Trp Tyr Tyr Thr His Met Arg Asp His 1220 1225 1230 Thr Asn Ser Asn Gly Cys Phe Trp Asn Phe Ile Ser Glu Glu His Gly 1235 1240 1245 Trp Gln Glu Lys 1250

53 AGATCTCGAT CCCGCGAAAT TAATACGACT CACTATAGGG GAATTGTGAG CGGATAACAA 60 TTCCCCTCTA GAAATAATTT TGTTTAACTT TAAGAAGGAG ATATACC ATG GGC CAT 116 Met Gly His 1 CAT CAT CAT CAT CAT CAT CAT CAT CAC AGC AGC GGC CAT ATC GAA GGT 164 His His His His His His His His His Ser Ser Gly His Ile Glu Gly 5 10 15 CGT CAT ATG GCT AGC ATG GCT CTT TCT TCT TAT ACA GAT GAT AAA ATT 212 Arg His Met Ala Ser Met Ala Leu Ser Ser Tyr Thr Asp Asp Lys Ile 20 25 30 35 TTA ATT TCA TAT TTT AAT AAG TTC TTT AAG AGA ATT AAA AGT AGT TCT 260 Leu Ile Ser Tyr Phe Asn Lys Phe Phe Lys Arg Ile Lys Ser Ser Ser 40 45 50 GTT TTA AAT ATG AGA TAT AAA AAT GAT AAA TAC GTA GAT ACT TCA GGA 308 Val Leu Asn Met Arg Tyr Lys Asn Asp Lys Tyr Val Asp Thr Ser Gly 55 60 65 TAT GAT TCA AAT ATA AAT ATT AAT GGA GAT GTA TAT AAA TAT CCA ACT 356 Tyr Asp Ser Asn Ile Asn Ile Asn Gly Asp Val Tyr Lys Tyr Pro Thr 70 75 80 AAT AAA AAT CAA TTT GGA ATA TAT AAT GAT AAA CTT AGT GAA GTT AAT 404 Asn Lys Asn Gln Phe Gly Ile Tyr Asn Asp Lys Leu Ser Glu Val Asn 85 90 95 ATA TCT CAA AAT GAT TAC ATT ATA TAT GAT AAT AAA TAT AAA AAT TTT 452 Ile Ser Gln Asn Asp Tyr Ile Ile Tyr Asp Asn Lys Tyr Lys Asn Phe 100 105 110 115 AGT ATT AGT TTT TGG GTA AGA ATT CCT AAC TAT GAT AAT AAG ATA GTA 500 Ser Ile Ser Phe Trp Val Arg Ile Pro Asn Tyr Asp Asn Lys Ile Val 120 125 130 AAT GTT AAT AAT GAA TAC ACT ATA ATA AAT TGT ATG AGG GAT AAT AAT 548 Asn Val Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Arg Asp Asn Asn 135 140 145 TCA GGA TGG AAA GTA TCT CTT AAT CAT AAT GAA ATA ATT TGG ACA TTG 596 Ser Gly Trp Lys Val Ser Leu Asn His Asn Glu Ile Ile Trp Thr Leu 150 155 160 CAA GAT AAT TCA GGA ATT AAT CAA AAA TTA GCA TTT AAC TAT GGT AAC 644 Gln Asp Asn Ser Gly Ile Asn Gln Lys Leu Ala Phe Asn Tyr Gly Asn 165 170 175 GCA AAT GGT ATT TCT GAT TAT ATA AAT AAG TGG ATT TTT GTA ACT ATA 692 Ala Asn Gly Ile Ser Asp Tyr Ile Asn Lys Trp Ile Phe Val Thr Ile 180 185 190 195 ACT AAT GAT AGA TTA GGA GAT TCT AAA CTT TAT ATT AAT GGA AAT TTA 740 Thr Asn Asp Arg Leu Gly Asp Ser Lys Leu Tyr Ile Asn Gly Asn Leu 200 205 210 ATA GAT AAA AAA TCA ATT TTA AAT TTA GGT AAT ATT CAT GTT AGT GAC 788 Ile Asp Lys Lys Ser Ile Leu Asn Leu Gly Asn Ile His Val Ser Asp 215 220 225 AAT ATA TTA TTT AAA ATA GTT AAT TGT AGT TAT ACA AGA TAT ATT GGT 836 Asn Ile Leu Phe Lys Ile Val Asn Cys Ser Tyr Thr Arg Tyr Ile Gly 230 235 240 ATT AGA TAT TTT AAT ATT TTT GAT AAA GAA TTA GAT GAA ACA GAA ATT 884 Ile Arg Tyr Phe Asn Ile Phe Asp Lys Glu Leu Asp Glu Thr Glu Ile 245 250 255 CAA ACT TTA TAT AAC AAT GAA CCT AAT GCA AAT ATT TTA AAG GAT TTT 932 Gln Thr Leu Tyr Asn Asn Glu Pro Asn Ala Asn Ile Leu Lys Asp Phe 260 265 270 275 TGG GGA AAT TAT TTG CTT TAT GAC AAA GAA TAC TAT TTA TTA AAT GTG 980 Trp Gly Asn Tyr Leu Leu Tyr Asp Lys Glu Tyr Tyr Leu Leu Asn Val 280 285 290 TTA AAA CCA AAT AAC TTT ATT AAT AGG AGA ACA GAT TCT ACT TTA AGC 1028 Leu Lys Pro Asn Asn Phe Ile Asn Arg Arg Thr Asp Ser Thr Leu Ser 295 300 305 ATT AAT AAT ATA AGA AGC ACT ATT CTT TTA GCT AAT AGA TTA TAT AGT 1076 Ile Asn Asn Ile Arg Ser Thr Ile Leu Leu Ala Asn Arg Leu Tyr Ser 310 315 320 GGA ATA AAA GTT AAA ATA CAA AGA GTT AAT AAT AGT AGT ACT AAC GAT 1124 Gly Ile Lys Val Lys Ile Gln Arg Val Asn Asn Ser Ser Thr Asn Asp 325 330 335 AAT CTT GTT AGA AAG AAT GAT CAG GTA TAT ATT AAT TTT GTA GCC AGC 1172 Asn Leu Val Arg Lys Asn Asp Gln Val Tyr Ile Asn Phe Val Ala Ser 340 345 350 355 AAA ACT CAC TTA CTT CCA TTA TAT GCT GAT ACA GCT ACC ACA AAT AAA 1220 Lys Thr His Leu Leu Pro Leu Tyr Ala Asp Thr Ala Thr Thr Asn Lys 360 365 370 GAG AAA ACA ATA AAA ATA TCA TCA TCT GGC AAT AGA TTT AAT CAA GTA 1268 Glu Lys Thr Ile Lys Ile Ser Ser Ser Gly Asn Arg Phe Asn Gln Val 375 380 385 GTA GTT ATG AAT TCA GTA GGA AAT TGT ACA ATG AAT TTT AAA AAT AAT 1316 Val Val Met Asn Ser Val Gly Asn Cys Thr Met Asn Phe Lys Asn Asn 390 395 400 AAT GGA AAT AAT ATT GGG TTG TTA GGT TTC AAG GCA GAT ACT GTA GTT 1364 Asn Gly Asn Asn Ile Gly Leu Leu Gly Phe Lys Ala Asp Thr Val Val 405 410 415 GCT AGT ACT TGG TAT TAT ACA CAT ATG AGA GAT AAT ACA AAC AGC AAT 1412 Ala Ser Thr Trp Tyr Tyr Thr His Met Arg Asp Asn Thr Asn Ser Asn 420 425 430 435 GGA TTT TTT TGG AAC TTT ATT TCT GAA GAA CAT GGA TGG CAA GAA AAA 1460 Gly Phe Phe Trp Asn Phe Ile Ser Glu Glu His Gly Trp Gln Glu Lys 440 445 450 TAA 1463

54 Met Gly His His His His His His His His His His Ser Ser Gly His 1 5 10 15 Ile Glu Gly Arg His Met Ala Ser Met Ala Leu Ser Ser Tyr Thr Asp 20 25 30 Asp Lys Ile Leu Ile Ser Tyr Phe Asn Lys Phe Phe Lys Arg Ile Lys 35 40 45 Ser Ser Ser Val Leu Asn Met Arg Tyr Lys Asn Asp Lys Tyr Val Asp 50 55 60 Thr Ser Gly Tyr Asp Ser Asn Ile Asn Ile Asn Gly Asp Val Tyr Lys 65 70 75 80 Tyr Pro Thr Asn Lys Asn Gln Phe Gly Ile Tyr Asn Asp Lys Leu Ser 85 90 95 Glu Val Asn Ile Ser Gln Asn Asp Tyr Ile Ile Tyr Asp Asn Lys Tyr 100 105 110 Lys Asn Phe Ser Ile Ser Phe Trp Val Arg Ile Pro Asn Tyr Asp Asn 115 120 125 Lys Ile Val Asn Val Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Arg 130 135 140 Asp Asn Asn Ser Gly Trp Lys Val Ser Leu Asn His Asn Glu Ile Ile 145 150 155 160 Trp Thr Leu Gln Asp Asn Ser Gly Ile Asn Gln Lys Leu Ala Phe Asn 165 170 175 Tyr Gly Asn Ala Asn Gly Ile Ser Asp Tyr Ile Asn Lys Trp Ile Phe 180 185 190 Val Thr Ile Thr Asn Asp Arg Leu Gly Asp Ser Lys Leu Tyr Ile Asn 195 200 205 Gly Asn Leu Ile Asp Lys Lys Ser Ile Leu Asn Leu Gly Asn Ile His 210 215 220 Val Ser Asp Asn Ile Leu Phe Lys Ile Val Asn Cys Ser Tyr Thr Arg 225 230 235 240 Tyr Ile Gly Ile Arg Tyr Phe Asn Ile Phe Asp Lys Glu Leu Asp Glu 245 250 255 Thr Glu Ile Gln Thr Leu Tyr Asn Asn Glu Pro Asn Ala Asn Ile Leu 260 265 270 Lys Asp Phe Trp Gly Asn Tyr Leu Leu Tyr Asp Lys Glu Tyr Tyr Leu 275 280 285 Leu Asn Val Leu Lys Pro Asn Asn Phe Ile Asn Arg Arg Thr Asp Ser 290 295 300 Thr Leu Ser Ile Asn Asn Ile Arg Ser Thr Ile Leu Leu Ala Asn Arg 305 310 315 320 Leu Tyr Ser Gly Ile Lys Val Lys Ile Gln Arg Val Asn Asn Ser Ser 325 330 335 Thr Asn Asp Asn Leu Val Arg Lys Asn Asp Gln Val Tyr Ile Asn Phe 340 345 350 Val Ala Ser Lys Thr His Leu Leu Pro Leu Tyr Ala Asp Thr Ala Thr 355 360 365 Thr Asn Lys Glu Lys Thr Ile Lys Ile Ser Ser Ser Gly Asn Arg Phe 370 375 380 Asn Gln Val Val Val Met Asn Ser Val Gly Asn Cys Thr Met Asn Phe 385 390 395 400 Lys Asn Asn Asn Gly Asn Asn Ile Gly Leu Leu Gly Phe Lys Ala Asp 405 410 415 Thr Val Val Ala Ser Thr Trp Tyr Tyr Thr His Met Arg Asp Asn Thr 420 425 430 Asn Ser Asn Gly Phe Phe Trp Asn Phe Ile Ser Glu Glu His Gly Trp 435 440 445 Gln Glu Lys 450

55 AGATCTCGAT CCCGCGAAAT TAATACGACT CACTATAGGG GAATTGTGAG CGGATAACAA 60 TTCCCCTCTA GAAATAATTT TGTTTAACTT TAAGAAGGAG ATATACC ATG GGC CAT 116 Met Gly His 1 CAT CAT CAT CAT CAT CAT CAT CAT CAC AGC AGC GGC CAT ATC GAA GGT 164 His His His His His His His His His Ser Ser Gly His Ile Glu Gly 5 10 15 CGT CAT ATG GCT AGC ATG GCT CTT TCT TCT TAT ACA GAT GAT AAA ATT 212 Arg His Met Ala Ser Met Ala Leu Ser Ser Tyr Thr Asp Asp Lys Ile 20 25 30 35 TTA ATT TCA TAT TTT AAT AAA TTC TTT AAG AGA ATT AAA AGT AGT TCA 260 Leu Ile Ser Tyr Phe Asn Lys Phe Phe Lys Arg Ile Lys Ser Ser Ser 40 45 50 GTT TTA AAT ATG AGA TAT AAA AAT GAT AAA TAC GTA GAT ACT TCA GGA 308 Val Leu Asn Met Arg Tyr Lys Asn Asp Lys Tyr Val Asp Thr Ser Gly 55 60 65 TAT GAT TCA AAT ATA AAT ATT AAT GGA GAT GTA TAT AAA TAT CCA ACT 356 Tyr Asp Ser Asn Ile Asn Ile Asn Gly Asp Val Tyr Lys Tyr Pro Thr 70 75 80 AAT AAA AAT CAA TTT GGA ATA TAT AAT GAT AAA CTT AGT GAA GTT AAT 404 Asn Lys Asn Gln Phe Gly Ile Tyr Asn Asp Lys Leu Ser Glu Val Asn 85 90 95 ATA TCT CAA AAT GAT TAC ATT ATA TAT GAT AAT AAA TAT AAA AAT TTT 452 Ile Ser Gln Asn Asp Tyr Ile Ile Tyr Asp Asn Lys Tyr Lys Asn Phe 100 105 110 115 AGT ATT AGT TTT TGG GTA AGA ATT CCT AAC TAT GAT AAT AAG ATA GTA 500 Ser Ile Ser Phe Trp Val Arg Ile Pro Asn Tyr Asp Asn Lys Ile Val 120 125 130 AAT GTT AAT AAT GAA TAC ACT ATA ATA AAT TGT ATG AGA GAT AAT AAT 548 Asn Val Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Arg Asp Asn Asn 135 140 145 TCA GGA TGG AAA GTA TCT CTT AAT CAT AAT GAA ATA ATT TGG ACA TTG 596 Ser Gly Trp Lys Val Ser Leu Asn His Asn Glu Ile Ile Trp Thr Leu 150 155 160 CAA GAT AAT GCA GGA ATT AAT CAA AAA TTA GCA TTT AAC TAT GGT AAC 644 Gln Asp Asn Ala Gly Ile Asn Gln Lys Leu Ala Phe Asn Tyr Gly Asn 165 170 175 GCA AAT GGT ATT TCT GAT TAT ATA AAT AAG TGG ATT TTT GTA ACT ATA 692 Ala Asn Gly Ile Ser Asp Tyr Ile Asn Lys Trp Ile Phe Val Thr Ile 180 185 190 195 ACT AAT GAT AGA TTA GGA GAT TCT AAA CTT TAT ATT AAT GGA AAT TTA 740 Thr Asn Asp Arg Leu Gly Asp Ser Lys Leu Tyr Ile Asn Gly Asn Leu 200 205 210 ATA GAT CAA AAA TCA ATT TTA AAT TTA GGT AAT ATT CAT GTT AGT GAC 788 Ile Asp Gln Lys Ser Ile Leu Asn Leu Gly Asn Ile His Val Ser Asp 215 220 225 AAT ATA TTA TTT AAA ATA GTT AAT TGT AGT TAT ACA AGA TAT ATT GGT 836 Asn Ile Leu Phe Lys Ile Val Asn Cys Ser Tyr Thr Arg Tyr Ile Gly 230 235 240 ATT AGA TAT TTT AAT ATT TTT GAT AAA GAA TTA GAT GAA ACA GAA ATT 884 Ile Arg Tyr Phe Asn Ile Phe Asp Lys Glu Leu Asp Glu Thr Glu Ile 245 250 255 CAA ACT TTA TAT AGC AAT GAA CCT AAT ACA AAT ATT TTG AAG GAT TTT 932 Gln Thr Leu Tyr Ser Asn Glu Pro Asn Thr Asn Ile Leu Lys Asp Phe 260 265 270 275 TGG GGA AAT TAT TTG CTT TAT GAC AAA GAA TAC TAT TTA TTA AAT GTG 980 Trp Gly Asn Tyr Leu Leu Tyr Asp Lys Glu Tyr Tyr Leu Leu Asn Val 280 285 290 TTA AAA CCA AAT AAC TTT ATT GAT AGG AGA AAA GAT TCT ACT TTA AGC 1028 Leu Lys Pro Asn Asn Phe Ile Asp Arg Arg Lys Asp Ser Thr Leu Ser 295 300 305 ATT AAT AAT ATA AGA AGC ACT ATT CTT TTA GCT AAT AGA TTA TAT AGT 1076 Ile Asn Asn Ile Arg Ser Thr Ile Leu Leu Ala Asn Arg Leu Tyr Ser 310 315 320 GGA ATA AAA GTT AAA ATA CAA AGA GTT AAT AAT AGT AGT ACT AAC GAT 1124 Gly Ile Lys Val Lys Ile Gln Arg Val Asn Asn Ser Ser Thr Asn Asp 325 330 335 AAT CTT GTT AGA AAG AAT GAT CAG GTA TAT ATT AAT TTT GTA GCC AGC 1172 Asn Leu Val Arg Lys Asn Asp Gln Val Tyr Ile Asn Phe Val Ala Ser 340 345 350 355 AAA ACT CAC TTA TTT CCA TTA TAT GCT GAT ACA GCT ACC ACA AAT AAA 1220 Lys Thr His Leu Phe Pro Leu Tyr Ala Asp Thr Ala Thr Thr Asn Lys 360 365 370 GAG AAA ACA ATA AAA ATA TCA TCA TCT GGC AAT AGA TTT AAT CAA GTA 1268 Glu Lys Thr Ile Lys Ile Ser Ser Ser Gly Asn Arg Phe Asn Gln Val 375 380 385 GTA GTT ATG AAT TCA GTA GGA AAT AAT TGT ACA ATG AAT TTT AAA AAT 1316 Val Val Met Asn Ser Val Gly Asn Asn Cys Thr Met Asn Phe Lys Asn 390 395 400 AAT AAT GGA AAT AAT ATT GGG TTG TTA GGT TTC AAG GCA GAT ACT GTA 1364 Asn Asn Gly Asn Asn Ile Gly Leu Leu Gly Phe Lys Ala Asp Thr Val 405 410 415 GTT GCT AGT ACT TGG TAT TAT ACA CAT ATG AGA GAT CAT ACA AAC AGC 1412 Val Ala Ser Thr Trp Tyr Tyr Thr His Met Arg Asp His Thr Asn Ser 420 425 430 435 AAT GGA TGT TTT TGG AAC TTT ATT TCT GAA GAA CAT GGA TGG CAA GAA 1460 Asn Gly Cys Phe Trp Asn Phe Ile Ser Glu Glu His Gly Trp Gln Glu 440 445 450 AAA TAAAAGCTT 1472 Lys

56 Met Gly His His His His His His His His His His Ser Ser Gly His 1 5 10 15 Ile Glu Gly Arg His Met Ala Ser Met Ala Leu Ser Ser Tyr Thr Asp 20 25 30 Asp Lys Ile Leu Ile Ser Tyr Phe Asn Lys Phe Phe Lys Arg Ile Lys 35 40 45 Ser Ser Ser Val Leu Asn Met Arg Tyr Lys Asn Asp Lys Tyr Val Asp 50 55 60 Thr Ser Gly Tyr Asp Ser Asn Ile Asn Ile Asn Gly Asp Val Tyr Lys 65 70 75 80 Tyr Pro Thr Asn Lys Asn Gln Phe Gly Ile Tyr Asn Asp Lys Leu Ser 85 90 95 Glu Val Asn Ile Ser Gln Asn Asp Tyr Ile Ile Tyr Asp Asn Lys Tyr 100 105 110 Lys Asn Phe Ser Ile Ser Phe Trp Val Arg Ile Pro Asn Tyr Asp Asn 115 120 125 Lys Ile Val Asn Val Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Arg 130 135 140 Asp Asn Asn Ser Gly Trp Lys Val Ser Leu Asn His Asn Glu Ile Ile 145 150 155 160 Trp Thr Leu Gln Asp Asn Ala Gly Ile Asn Gln Lys Leu Ala Phe Asn 165 170 175 Tyr Gly Asn Ala Asn Gly Ile Ser Asp Tyr Ile Asn Lys Trp Ile Phe 180 185 190 Val Thr Ile Thr Asn Asp Arg Leu Gly Asp Ser Lys Leu Tyr Ile Asn 195 200 205 Gly Asn Leu Ile Asp Gln Lys Ser Ile Leu Asn Leu Gly Asn Ile His 210 215 220 Val Ser Asp Asn Ile Leu Phe Lys Ile Val Asn Cys Ser Tyr Thr Arg 225 230 235 240 Tyr Ile Gly Ile Arg Tyr Phe Asn Ile Phe Asp Lys Glu Leu Asp Glu 245 250 255 Thr Glu Ile Gln Thr Leu Tyr Ser Asn Glu Pro Asn Thr Asn Ile Leu 260 265 270 Lys Asp Phe Trp Gly Asn Tyr Leu Leu Tyr Asp Lys Glu Tyr Tyr Leu 275 280 285 Leu Asn Val Leu Lys Pro Asn Asn Phe Ile Asp Arg Arg Lys Asp Ser 290 295 300 Thr Leu Ser Ile Asn Asn Ile Arg Ser Thr Ile Leu Leu Ala Asn Arg 305 310 315 320 Leu Tyr Ser Gly Ile Lys Val Lys Ile Gln Arg Val Asn Asn Ser Ser 325 330 335 Thr Asn Asp Asn Leu Val Arg Lys Asn Asp Gln Val Tyr Ile Asn Phe 340 345 350 Val Ala Ser Lys Thr His Leu Phe Pro Leu Tyr Ala Asp Thr Ala Thr 355 360 365 Thr Asn Lys Glu Lys Thr Ile Lys Ile Ser Ser Ser Gly Asn Arg Phe 370 375 380 Asn Gln Val Val Val Met Asn Ser Val Gly Asn Asn Cys Thr Met Asn 385 390 395 400 Phe Lys Asn Asn Asn Gly Asn Asn Ile Gly Leu Leu Gly Phe Lys Ala 405 410 415 Asp Thr Val Val Ala Ser Thr Trp Tyr Tyr Thr His Met Arg Asp His 420 425 430 Thr Asn Ser Asn Gly Cys Phe Trp Asn Phe Ile Ser Glu Glu His Gly 435 440 445 Trp Gln Glu Lys 450

57 CGCCATGGCT CTTTCTTCTT ATACAGATGA T 31

58 GCAAGCTTTT ATTTTTCTTG CCATCCATG 29

59 ATG CCA ATA ACA ATT AAC AAC TTT AAT TAT TCA GAT CCT GTT GAT AAT 48 Met Pro Ile Thr Ile Asn Asn Phe Asn Tyr Ser Asp Pro Val Asp Asn 1 5 10 15 AAA AAT ATT TTA TAT TTA GAT ACT CAT TTA AAT ACA CTA GCT AAT GAG 96 Lys Asn Ile Leu Tyr Leu Asp Thr His Leu Asn Thr Leu Ala Asn Glu 20 25 30 CCT GAA AAA GCC TTT CGC ATT ACA GGA AAT ATA TGG GTA ATA CCT GAT 144 Pro Glu Lys Ala Phe Arg Ile Thr Gly Asn Ile Trp Val Ile Pro Asp 35 40 45 AGA TTT TCA AGA AAT TCT AAT CCA AAT TTA AAT AAA CCT CCT CGA GTT 192 Arg Phe Ser Arg Asn Ser Asn Pro Asn Leu Asn Lys Pro Pro Arg Val 50 55 60 ACA AGC CCT AAA AGT GGT TAT TAT GAT CCT AAT TAT TTG AGT ACT GAT 240 Thr Ser Pro Lys Ser Gly Tyr Tyr Asp Pro Asn Tyr Leu Ser Thr Asp 65 70 75 80 TCT GAC AAA GAT ACA TTT TTA AAA GAA ATT ATA AAG TTA TTT AAA AGA 288 Ser Asp Lys Asp Thr Phe Leu Lys Glu Ile Ile Lys Leu Phe Lys Arg 85 90 95 ATT AAT TCT AGA GAA ATA GGA GAA GAA TTA ATA TAT AGA CTT TCG ACA 336 Ile Asn Ser Arg Glu Ile Gly Glu Glu Leu Ile Tyr Arg Leu Ser Thr 100 105 110 GAT ATA CCC TTT CCT GGG AAT AAC AAT ACT CCA ATT AAT ACT TTT GAT 384 Asp Ile Pro Phe Pro Gly Asn Asn Asn Thr Pro Ile Asn Thr Phe Asp 115 120 125 TTT GAT GTA GAT TTT AAC AGT GTT GAT GTT AAA ACT AGA CAA GGT AAC 432 Phe Asp Val Asp Phe Asn Ser Val Asp Val Lys Thr Arg Gln Gly Asn 130 135 140 AAC TGG GTT AAA ACT GGT AGC ATA AAT CCT AGT GTT ATA ATA ACT GGA 480 Asn Trp Val Lys Thr Gly Ser Ile Asn Pro Ser Val Ile Ile Thr Gly 145 150 155 160 CCT AGA GAA AAC ATT ATA GAT CCA GAA ACT TCT ACG TTT AAA TTA ACT 528 Pro Arg Glu Asn Ile Ile Asp Pro Glu Thr Ser Thr Phe Lys Leu Thr 165 170 175 AAC AAT ACT TTT GCG GCA CAA GAA GGA TTT GGT GCT TTA TCA ATA ATT 576 Asn Asn Thr Phe Ala Ala Gln Glu Gly Phe Gly Ala Leu Ser Ile Ile 180 185 190 TCA ATA TCA CCT AGA TTT ATG CTA ACA TAT AGT AAT GCA ACT AAT GAT 624 Ser Ile Ser Pro Arg Phe Met Leu Thr Tyr Ser Asn Ala Thr Asn Asp 195 200 205 GTA GGA GAG GGT AGA TTT TCT AAG TCT GAA TTT TGC ATG GAT CCA ATA 672 Val Gly Glu Gly Arg Phe Ser Lys Ser Glu Phe Cys Met Asp Pro Ile 210 215 220 CTA ATT TTA ATG CAT GAA CTT AAT CAT GCA ATG CAT AAT TTA TAT GGA 720 Leu Ile Leu Met His Glu Leu Asn His Ala Met His Asn Leu Tyr Gly 225 230 235 240 ATA GCT ATA CCA AAT GAT CAA ACA ATT TCA TCT GTA ACT AGT AAT ATT 768 Ile Ala Ile Pro Asn Asp Gln Thr Ile Ser Ser Val Thr Ser Asn Ile 245 250 255 TTT TAT TCT CAA TAT AAT GTG AAA TTA GAG TAT GCA GAA ATA TAT GCA 816 Phe Tyr Ser Gln Tyr Asn Val Lys Leu Glu Tyr Ala Glu Ile Tyr Ala 260 265 270 TTT GGA GGT CCA ACT ATA GAC CTT ATT CCT AAA AGT GCA AGG AAA TAT 864 Phe Gly Gly Pro Thr Ile Asp Leu Ile Pro Lys Ser Ala Arg Lys Tyr 275 280 285 TTT GAG GAA AAG GCA TTG GAT TAT TAT AGA TCT ATA GCT AAA AGA CTT 912 Phe Glu Glu Lys Ala Leu Asp Tyr Tyr Arg Ser Ile Ala Lys Arg Leu 290 295 300 AAT AGT ATA ACT ACT GCA AAT CCT TCA AGC TTT AAT AAA TAT ATA GGG 960 Asn Ser Ile Thr Thr Ala Asn Pro Ser Ser Phe Asn Lys Tyr Ile Gly 305 310 315 320 GAA TAT AAA CAG AAA CTT ATT AGA AAG TAT AGA TTC GTA GTA GAA TCT 1008 Glu Tyr Lys Gln Lys Leu Ile Arg Lys Tyr Arg Phe Val Val Glu Ser 325 330 335 TCA GGT GAA GTT ACA GTA AAT CGT AAT AAG TTT GTT GAG TTA TAT AAT 1056 Ser Gly Glu Val Thr Val Asn Arg Asn Lys Phe Val Glu Leu Tyr Asn 340 345 350 GAA CTT ACA CAA ATA TTT ACA GAA TTT AAC TAC GCT AAA ATA TAT AAT 1104 Glu Leu Thr Gln Ile Phe Thr Glu Phe Asn Tyr Ala Lys Ile Tyr Asn 355 360 365 GTA CAA AAT AGG AAA ATA TAT CTT TCA AAT GTA TAT ACT CCG GTT ACG 1152 Val Gln Asn Arg Lys Ile Tyr Leu Ser Asn Val Tyr Thr Pro Val Thr 370 375 380 GCG AAT ATA TTA GAC GAT AAT GTT TAT GAT ATA CAA AAT GGA TTT AAT 1200 Ala Asn Ile Leu Asp Asp Asn Val Tyr Asp Ile Gln Asn Gly Phe Asn 385 390 395 400 ATA CCT AAA AGT AAT TTA AAT GTA CTA TTT ATG GGT CAA AAT TTA TCT 1248 Ile Pro Lys Ser Asn Leu Asn Val Leu Phe Met Gly Gln Asn Leu Ser 405 410 415 CGA AAT CCA GCA TTA AGA AAA GTC AAT CCT GAA AAT ATG CTT TAT TTA 1296 Arg Asn Pro Ala Leu Arg Lys Val Asn Pro Glu Asn Met Leu Tyr Leu 420 425 430 TTT ACA AAA TTT TGT CAT AAA GCA ATA GAT GGT AGA TCA TTA TAT AAT 1344 Phe Thr Lys Phe Cys His Lys Ala Ile Asp Gly Arg Ser Leu Tyr Asn 435 440 445 AAA ACA TTA GAT TGT AGA GAG CTT TTA GTT AAA AAT ACT GAC TTA CCC 1392 Lys Thr Leu Asp Cys Arg Glu Leu Leu Val Lys Asn Thr Asp Leu Pro 450 455 460 TTT ATA GGT GAT ATT AGT GAT GTT AAA ACT GAT ATA TTT TTA AGA AAA 1440 Phe Ile Gly Asp Ile Ser Asp Val Lys Thr Asp Ile Phe Leu Arg Lys 465 470 475 480 GAT ATT AAT GAA GAA ACT GAA GTT ATA TAC TAT CCG GAC AAT GTT TCA 1488 Asp Ile Asn Glu Glu Thr Glu Val Ile Tyr Tyr Pro Asp Asn Val Ser 485 490 495 GTA GAT CAA GTT ATT CTC AGT AAG AAT ACC TCA GAA CAT GGA CAA CTA 1536 Val Asp Gln Val Ile Leu Ser Lys Asn Thr Ser Glu His Gly Gln Leu 500 505 510 GAT TTA TTA TAC CCT AGT ATT GAC AGT GAG AGT GAA ATA TTA CCA GGG 1584 Asp Leu Leu Tyr Pro Ser Ile Asp Ser Glu Ser Glu Ile Leu Pro Gly 515 520 525 GAG AAT CAA GTC TTT TAT GAT AAT AGA ACT CAA AAT GTT GAT TAT TTG 1632 Glu Asn Gln Val Phe Tyr Asp Asn Arg Thr Gln Asn Val Asp Tyr Leu 530 535 540 AAT TCT TAT TAT TAC CTA GAA TCT CAA AAA CTA AGT GAT AAT GTT GAA 1680 Asn Ser Tyr Tyr Tyr Leu Glu Ser Gln Lys Leu Ser Asp Asn Val Glu 545 550 555 560 GAT TTT ACT TTT ACG AGA TCA ATT GAG GAG GCT TTG GAT AAT AGT GCA 1728 Asp Phe Thr Phe Thr Arg Ser Ile Glu Glu Ala Leu Asp Asn Ser Ala 565 570 575 AAA GTA TAT ACT TAC TTT CCT ACA CTA GCT AAT AAA GTA AAT GCG GGT 1776 Lys Val Tyr Thr Tyr Phe Pro Thr Leu Ala Asn Lys Val Asn Ala Gly 580 585 590 GTT CAA GGT GGT TTA TTT TTA ATG TGG GCA AAT GAT GTA GTT GAA GAT 1824 Val Gln Gly Gly Leu Phe Leu Met Trp Ala Asn Asp Val Val Glu Asp 595 600 605 TTT ACT ACA AAT ATT CTA AGA AAA GAT ACA TTA GAT AAA ATA TCA GAT 1872 Phe Thr Thr Asn Ile Leu Arg Lys Asp Thr Leu Asp Lys Ile Ser Asp 610 615 620 GTA TCA GCT ATT ATT CCC TAT ATA GGA CCC GCA TTA AAT ATA AGT AAT 1920 Val Ser Ala Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Ser Asn 625 630 635 640 TCT GTA AGA AGA GGA AAT TTT ACT GAA GCA TTT GCA GTT ACT GGT GTA 1968 Ser Val Arg Arg Gly Asn Phe Thr Glu Ala Phe Ala Val Thr Gly Val 645 650 655 ACT ATT TTA TTA GAA GCA TTT CCT GAA TTT ACA ATA CCT GCA CTT GGT 2016 Thr Ile Leu Leu Glu Ala Phe Pro Glu Phe Thr Ile Pro Ala Leu Gly 660 665 670 GCA TTT GTG ATT TAT AGT AAG GTT CAA GAA AGA AAC GAG ATT ATT AAA 2064 Ala Phe Val Ile Tyr Ser Lys Val Gln Glu Arg Asn Glu Ile Ile Lys 675 680 685 ACT ATA GAT AAT TGT TTA GAA CAA AGG ATT AAG AGA TGG AAA GAT TCA 2112 Thr Ile Asp Asn Cys Leu Glu Gln Arg Ile Lys Arg Trp Lys Asp Ser 690 695 700 TAT GAA TGG ATG ATG GGA ACG TGG TTA TCC AGG ATT ATT ACT CAA TTT 2160 Tyr Glu Trp Met Met Gly Thr Trp Leu Ser Arg Ile Ile Thr Gln Phe 705 710 715 720 AAT AAT ATA AGT TAT CAA ATG TAT GAT TCT TTA AAT TAT CAG GCA GGT 2208 Asn Asn Ile Ser Tyr Gln Met Tyr Asp Ser Leu Asn Tyr Gln Ala Gly 725 730 735 GCA ATC AAA GCT AAA ATA GAT TTA GAA TAT AAA AAA TAT TCA GGA AGT 2256 Ala Ile Lys Ala Lys Ile Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser 740 745 750 GAT AAA GAA AAT ATA AAA AGT CAA GTT GAA AAT TTA AAA AAT AGT TTA 2304 Asp Lys Glu Asn Ile Lys Ser Gln Val Glu Asn Leu Lys Asn Ser Leu 755 760 765 GAT GTA AAA ATT TCG GAA GCA ATG AAT AAT ATA AAT AAA TTT ATA CGA 2352 Asp Val Lys Ile Ser Glu Ala Met Asn Asn Ile Asn Lys Phe Ile Arg 770 775 780 GAA TGT TCC GTA ACA TAT TTA TTT AAA AAT ATG TTA CCT AAA GTA ATT 2400 Glu Cys Ser Val Thr Tyr Leu Phe Lys Asn Met Leu Pro Lys Val Ile 785 790 795 800 GAT GAA TTA AAT GAG TTT GAT CGA AAT ACT AAA GCA AAA TTA ATT AAT 2448 Asp Glu Leu Asn Glu Phe Asp Arg Asn Thr Lys Ala Lys Leu Ile Asn 805 810 815 CTT ATA GAT AGT CAT AAT ATT ATT CTA GTT GGT GAA GTA GAT AAA TTA 2496 Leu Ile Asp Ser His Asn Ile Ile Leu Val Gly Glu Val Asp Lys Leu 820 825 830 AAA GCA AAA GTA AAT AAT AGC TTT CAA AAT ACA ATA CCC TTT AAT ATT 2544 Lys Ala Lys Val Asn Asn Ser Phe Gln Asn Thr Ile Pro Phe Asn Ile 835 840 845 TTT TCA TAT ACT AAT AAT TCT TTA TTA AAA GAT ATA ATT AAT GAA TAT 2592 Phe Ser Tyr Thr Asn Asn Ser Leu Leu Lys Asp Ile Ile Asn Glu Tyr 850 855 860 TTC AAT AAT ATT AAT GAT TCA AAA ATT TTG AGC CTA CAA AAC AGA AAA 2640 Phe Asn Asn Ile Asn Asp Ser Lys Ile Leu Ser Leu Gln Asn Arg Lys 865 870 875 880 AAT ACT TTA GTG GAT ACA TCA GGA TAT AAT GCA GAA GTG AGT GAA GAA 2688 Asn Thr Leu Val Asp Thr Ser Gly Tyr Asn Ala Glu Val Ser Glu Glu 885 890 895 GGC GAT GTT CAG CTT AAT CCA ATA TTT CCA TTT GAC TTT AAA TTA GGT 2736 Gly Asp Val Gln Leu Asn Pro Ile Phe Pro Phe Asp Phe Lys Leu Gly 900 905 910 AGT TCA GGG GAG GAT AGA GGT AAA GTT ATA GTA ACC CAG AAT GAA AAT 2784 Ser Ser Gly Glu Asp Arg Gly Lys Val Ile Val Thr Gln Asn Glu Asn 915 920 925 ATT GTA TAT AAT TCT ATG TAT GAA AGT TTT AGC ATT AGT TTT TGG ATT 2832 Ile Val Tyr Asn Ser Met Tyr Glu Ser Phe Ser Ile Ser Phe Trp Ile 930 935 940 AGA ATA AAT AAA TGG GTA AGT AAT TTA CCT GGA TAT ACT ATA ATT GAT 2880 Arg Ile Asn Lys Trp Val Ser Asn Leu Pro Gly Tyr Thr Ile Ile Asp 945 950 955 960 AGT GTT AAA AAT AAC TCA GGT TGG AGT ATA GGT ATT ATT AGT AAT TTT 2928 Ser Val Lys Asn Asn Ser Gly Trp Ser Ile Gly Ile Ile Ser Asn Phe 965 970 975 TTA GTA TTT ACT TTA AAA CAA AAT GAA GAT AGT GAA CAA AGT ATA AAT 2976 Leu Val Phe Thr Leu Lys Gln Asn Glu Asp Ser Glu Gln Ser Ile Asn 980 985 990 TTT AGT TAT GAT ATA TCA AAT AAT GCT CCT GGA TAC AAT AAA TGG TTT 3024 Phe Ser Tyr Asp Ile Ser Asn Asn Ala Pro Gly Tyr Asn Lys Trp Phe 995 1000 1005 TTT GTA ACT GTT ACT AAC AAT ATG ATG GGA AAT ATG AAG ATT TAT ATA 3072 Phe Val Thr Val Thr Asn Asn Met Met Gly Asn Met Lys Ile Tyr Ile 1010 1015 1020 AAT GGA AAA TTA ATA GAT ACT ATA AAA GTT AAA GAA CTA ACT GGA ATT 3120 Asn Gly Lys Leu Ile Asp Thr Ile Lys Val Lys Glu Leu Thr Gly Ile 1025 1030 1035 1040 AAT TTT AGC AAA ACT ATA ACA TTT GAA ATA AAT AAA ATT CCA GAT ACC 3168 Asn Phe Ser Lys Thr Ile Thr Phe Glu Ile Asn Lys Ile Pro Asp Thr 1045 1050 1055 GGT TTG ATT ACT TCA GAT TCT GAT AAC ATC AAT ATG TGG ATA AGA GAT 3216 Gly Leu Ile Thr Ser Asp Ser Asp Asn Ile Asn Met Trp Ile Arg Asp 1060 1065 1070 TTT TAT ATA TTT GCT AAA GAA TTA GAT GGT AAA GAT ATT AAT ATA TTA 3264 Phe Tyr Ile Phe Ala Lys Glu Leu Asp Gly Lys Asp Ile Asn Ile Leu 1075 1080 1085 TTT AAT AGC TTG CAA TAT ACT AAT GTT GTA AAA GAT TAT TGG GGA AAT 3312 Phe Asn Ser Leu Gln Tyr Thr Asn Val Val Lys Asp Tyr Trp Gly Asn 1090 1095 1100 GAT TTA AGA TAT AAT AAA GAA TAT TAT ATG GTT AAT ATA GAT TAT TTA 3360 Asp Leu Arg Tyr Asn Lys Glu Tyr Tyr Met Val Asn Ile Asp Tyr Leu 1105 1110 1115 1120 AAT AGA TAT ATG TAT GCG AAC TCA CGA CAA ATT GTT TTT AAT ACA CGT 3408 Asn Arg Tyr Met Tyr Ala Asn Ser Arg Gln Ile Val Phe Asn Thr Arg 1125 1130 1135 AGA AAT AAT AAT GAC TTC AAT GAA GGA TAT AAA ATT ATA ATA AAA AGA 3456 Arg Asn Asn Asn Asp Phe Asn Glu Gly Tyr Lys Ile Ile Ile Lys Arg 1140 1145 1150 ATC AGA GGA AAT ACA AAT GAT ACT AGA GTA CGA GGA GGA GAT ATT TTA 3504 Ile Arg Gly Asn Thr Asn Asp Thr Arg Val Arg Gly Gly Asp Ile Leu 1155 1160 1165 TAT TTT GAT ATG ACA ATT AAT AAC AAA GCA TAT AAT TTG TTT ATG AAG 3552 Tyr Phe Asp Met Thr Ile Asn Asn Lys Ala Tyr Asn Leu Phe Met Lys 1170 1175 1180 AAT GAA ACT ATG TAT GCA GAT AAT CAT AGT ACT GAA GAT ATA TAT GCT 3600 Asn Glu Thr Met Tyr Ala Asp Asn His Ser Thr Glu Asp Ile Tyr Ala 1185 1190 1195 1200 ATA GGT TTA AGA GAA CAA ACA AAG GAT ATA AAT GAT AAT ATT ATA TTT 3648 Ile Gly Leu Arg Glu Gln Thr Lys Asp Ile Asn Asp Asn Ile Ile Phe 1205 1210 1215 CAA ATA CAA CCA ATG AAT AAT ACT TAT TAT TAC GCA TCT CAA ATA TTT 3696 Gln Ile Gln Pro Met Asn Asn Thr Tyr Tyr Tyr Ala Ser Gln Ile Phe 1220 1225 1230 AAA TCA AAT TTT AAT GGA GAA AAT ATT TCT GGA ATA TGT TCA ATA GGT 3744 Lys Ser Asn Phe Asn Gly Glu Asn Ile Ser Gly Ile Cys Ser Ile Gly 1235 1240 1245 ACT TAT CGT TTT AGA CTT GGA GGT GAT TGG TAT AGA CAC AAT TAT TTG 3792 Thr Tyr Arg Phe Arg Leu Gly Gly Asp Trp Tyr Arg His Asn Tyr Leu 1250 1255 1260 GTG CCT ACT GTG AAG CAA GGA AAT TAT GCT TCA TTA TTA GAA TCA ACA 3840 Val Pro Thr Val Lys Gln Gly Asn Tyr Ala Ser Leu Leu Glu Ser Thr 1265 1270 1275 1280 TCA ACT CAT TGG GGT TTT GTA CCT GTA AGT GAA TAA 3876 Ser Thr His Trp Gly Phe Val Pro Val Ser Glu 1285 1290

60 Met Pro Ile Thr Ile Asn Asn Phe Asn Tyr Ser Asp Pro Val Asp Asn 1 5 10 15 Lys Asn Ile Leu Tyr Leu Asp Thr His Leu Asn Thr Leu Ala Asn Glu 20 25 30 Pro Glu Lys Ala Phe Arg Ile Thr Gly Asn Ile Trp Val Ile Pro Asp 35 40 45 Arg Phe Ser Arg Asn Ser Asn Pro Asn Leu Asn Lys Pro Pro Arg Val 50 55 60 Thr Ser Pro Lys Ser Gly Tyr Tyr Asp Pro Asn Tyr Leu Ser Thr Asp 65 70 75 80 Ser Asp Lys Asp Thr Phe Leu Lys Glu Ile Ile Lys Leu Phe Lys Arg 85 90 95 Ile Asn Ser Arg Glu Ile Gly Glu Glu Leu Ile Tyr Arg Leu Ser Thr 100 105 110 Asp Ile Pro Phe Pro Gly Asn Asn Asn Thr Pro Ile Asn Thr Phe Asp 115 120 125 Phe Asp Val Asp Phe Asn Ser Val Asp Val Lys Thr Arg Gln Gly Asn 130 135 140 Asn Trp Val Lys Thr Gly Ser Ile Asn Pro Ser Val Ile Ile Thr Gly 145 150 155 160 Pro Arg Glu Asn Ile Ile Asp Pro Glu Thr Ser Thr Phe Lys Leu Thr 165 170 175 Asn Asn Thr Phe Ala Ala Gln Glu Gly Phe Gly Ala Leu Ser Ile Ile 180 185 190 Ser Ile Ser Pro Arg Phe Met Leu Thr Tyr Ser Asn Ala Thr Asn Asp 195 200 205 Val Gly Glu Gly Arg Phe Ser Lys Ser Glu Phe Cys Met Asp Pro Ile 210 215 220 Leu Ile Leu Met His Glu Leu Asn His Ala Met His Asn Leu Tyr Gly 225 230 235 240 Ile Ala Ile Pro Asn Asp Gln Thr Ile Ser Ser Val Thr Ser Asn Ile 245 250 255 Phe Tyr Ser Gln Tyr Asn Val Lys Leu Glu Tyr Ala Glu Ile Tyr Ala 260 265 270 Phe Gly Gly Pro Thr Ile Asp Leu Ile Pro Lys Ser Ala Arg Lys Tyr 275 280 285 Phe Glu Glu Lys Ala Leu Asp Tyr Tyr Arg Ser Ile Ala Lys Arg Leu 290 295 300 Asn Ser Ile Thr Thr Ala Asn Pro Ser Ser Phe Asn Lys Tyr Ile Gly 305 310 315 320 Glu Tyr Lys Gln Lys Leu Ile Arg Lys Tyr Arg Phe Val Val Glu Ser 325 330 335 Ser Gly Glu Val Thr Val Asn Arg Asn Lys Phe Val Glu Leu Tyr Asn 340 345 350 Glu Leu Thr Gln Ile Phe Thr Glu Phe Asn Tyr Ala Lys Ile Tyr Asn 355 360 365 Val Gln Asn Arg Lys Ile Tyr Leu Ser Asn Val Tyr Thr Pro Val Thr 370 375 380 Ala Asn Ile Leu Asp Asp Asn Val Tyr Asp Ile Gln Asn Gly Phe Asn 385 390 395 400 Ile Pro Lys Ser Asn Leu Asn Val Leu Phe Met Gly Gln Asn Leu Ser 405 410 415 Arg Asn Pro Ala Leu Arg Lys Val Asn Pro Glu Asn Met Leu Tyr Leu 420 425 430 Phe Thr Lys Phe Cys His Lys Ala Ile Asp Gly Arg Ser Leu Tyr Asn 435 440 445 Lys Thr Leu Asp Cys Arg Glu Leu Leu Val Lys Asn Thr Asp Leu Pro 450 455 460 Phe Ile Gly Asp Ile Ser Asp Val Lys Thr Asp Ile Phe Leu Arg Lys 465 470 475 480 Asp Ile Asn Glu Glu Thr Glu Val Ile Tyr Tyr Pro Asp Asn Val Ser 485 490 495 Val Asp Gln Val Ile Leu Ser Lys Asn Thr Ser Glu His Gly Gln Leu 500 505 510 Asp Leu Leu Tyr Pro Ser Ile Asp Ser Glu Ser Glu Ile Leu Pro Gly 515 520 525 Glu Asn Gln Val Phe Tyr Asp Asn Arg Thr Gln Asn Val Asp Tyr Leu 530 535 540 Asn Ser Tyr Tyr Tyr Leu Glu Ser Gln Lys Leu Ser Asp Asn Val Glu 545 550 555 560 Asp Phe Thr Phe Thr Arg Ser Ile Glu Glu Ala Leu Asp Asn Ser Ala 565 570 575 Lys Val Tyr Thr Tyr Phe Pro Thr Leu Ala Asn Lys Val Asn Ala Gly 580 585 590 Val Gln Gly Gly Leu Phe Leu Met Trp Ala Asn Asp Val Val Glu Asp 595 600 605 Phe Thr Thr Asn Ile Leu Arg Lys Asp Thr Leu Asp Lys Ile Ser Asp 610 615 620 Val Ser Ala Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Ser Asn 625 630 635 640 Ser Val Arg Arg Gly Asn Phe Thr Glu Ala Phe Ala Val Thr Gly Val 645 650 655 Thr Ile Leu Leu Glu Ala Phe Pro Glu Phe Thr Ile Pro Ala Leu Gly 660 665 670 Ala Phe Val Ile Tyr Ser Lys Val Gln Glu Arg Asn Glu Ile Ile Lys 675 680 685 Thr Ile Asp Asn Cys Leu Glu Gln Arg Ile Lys Arg Trp Lys Asp Ser 690 695 700 Tyr Glu Trp Met Met Gly Thr Trp Leu Ser Arg Ile Ile Thr Gln Phe 705 710 715 720 Asn Asn Ile Ser Tyr Gln Met Tyr Asp Ser Leu Asn Tyr Gln Ala Gly 725 730 735 Ala Ile Lys Ala Lys Ile Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser 740 745 750 Asp Lys Glu Asn Ile Lys Ser Gln Val Glu Asn Leu Lys Asn Ser Leu 755 760 765 Asp Val Lys Ile Ser Glu Ala Met Asn Asn Ile Asn Lys Phe Ile Arg 770 775 780 Glu Cys Ser Val Thr Tyr Leu Phe Lys Asn Met Leu Pro Lys Val Ile 785 790 795 800 Asp Glu Leu Asn Glu Phe Asp Arg Asn Thr Lys Ala Lys Leu Ile Asn 805 810 815 Leu Ile Asp Ser His Asn Ile Ile Leu Val Gly Glu Val Asp Lys Leu 820 825 830 Lys Ala Lys Val Asn Asn Ser Phe Gln Asn Thr Ile Pro Phe Asn Ile 835 840 845 Phe Ser Tyr Thr Asn Asn Ser Leu Leu Lys Asp Ile Ile Asn Glu Tyr 850 855 860 Phe Asn Asn Ile Asn Asp Ser Lys Ile Leu Ser Leu Gln Asn Arg Lys 865 870 875 880 Asn Thr Leu Val Asp Thr Ser Gly Tyr Asn Ala Glu Val Ser Glu Glu 885 890 895 Gly Asp Val Gln Leu Asn Pro Ile Phe Pro Phe Asp Phe Lys Leu Gly 900 905 910 Ser Ser Gly Glu Asp Arg Gly Lys Val Ile Val Thr Gln Asn Glu Asn 915 920 925 Ile Val Tyr Asn Ser Met Tyr Glu Ser Phe Ser Ile Ser Phe Trp Ile 930 935 940 Arg Ile Asn Lys Trp Val Ser Asn Leu Pro Gly Tyr Thr Ile Ile Asp 945 950 955 960 Ser Val Lys Asn Asn Ser Gly Trp Ser Ile Gly Ile Ile Ser Asn Phe 965 970 975 Leu Val Phe Thr Leu Lys Gln Asn Glu Asp Ser Glu Gln Ser Ile Asn 980 985 990 Phe Ser Tyr Asp Ile Ser Asn Asn Ala Pro Gly Tyr Asn Lys Trp Phe 995 1000 1005 Phe Val Thr Val Thr Asn Asn Met Met Gly Asn Met Lys Ile Tyr Ile 1010 1015 1020 Asn Gly Lys Leu Ile Asp Thr Ile Lys Val Lys Glu Leu Thr Gly Ile 1025 1030 1035 1040 Asn Phe Ser Lys Thr Ile Thr Phe Glu Ile Asn Lys Ile Pro Asp Thr 1045 1050 1055 Gly Leu Ile Thr Ser Asp Ser Asp Asn Ile Asn Met Trp Ile Arg Asp 1060 1065 1070 Phe Tyr Ile Phe Ala Lys Glu Leu Asp Gly Lys Asp Ile Asn Ile Leu 1075 1080 1085 Phe Asn Ser Leu Gln Tyr Thr Asn Val Val Lys Asp Tyr Trp Gly Asn 1090 1095 1100 Asp Leu Arg Tyr Asn Lys Glu Tyr Tyr Met Val Asn Ile Asp Tyr Leu 1105 1110 1115 1120 Asn Arg Tyr Met Tyr Ala Asn Ser Arg Gln Ile Val Phe Asn Thr Arg 1125 1130 1135 Arg Asn Asn Asn Asp Phe Asn Glu Gly Tyr Lys Ile Ile Ile Lys Arg 1140 1145 1150 Ile Arg Gly Asn Thr Asn Asp Thr Arg Val Arg Gly Gly Asp Ile Leu 1155 1160 1165 Tyr Phe Asp Met Thr Ile Asn Asn Lys Ala Tyr Asn Leu Phe Met Lys 1170 1175 1180 Asn Glu Thr Met Tyr Ala Asp Asn His Ser Thr Glu Asp Ile Tyr Ala 1185 1190 1195 1200 Ile Gly Leu Arg Glu Gln Thr Lys Asp Ile Asn Asp Asn Ile Ile Phe 1205 1210 1215 Gln Ile Gln Pro Met Asn Asn Thr Tyr Tyr Tyr Ala Ser Gln Ile Phe 1220 1225 1230 Lys Ser Asn Phe Asn Gly Glu Asn Ile Ser Gly Ile Cys Ser Ile Gly 1235 1240 1245 Thr Tyr Arg Phe Arg Leu Gly Gly Asp Trp Tyr Arg His Asn Tyr Leu 1250 1255 1260 Val Pro Thr Val Lys Gln Gly Asn Tyr Ala Ser Leu Leu Glu Ser Thr 1265 1270 1275 1280 Ser Thr His Trp Gly Phe Val Pro Val Ser Glu 1285 1290

61 AGATCTCGAT CCCGCGAAAT TAATACGACT CACTATAGGG GAATTGTGAGCGGATAACAA 60 TTCCCCTCTA GAAATAATTT TGTTTAACTT TAAGAAGGAG ATATACC ATG GGC CAT 116 Met Gly His 1 CAT CAT CAT CAT CAT CAT CAT CAT CAC AGC AGC GGC CAT ATC GAA GGT 164 His His His His His His His His His Ser Ser Gly His Ile Glu Gly 5 10 15 CGT CAT ATG GCT AGC ATG GCT TTA TTA AAA GAT ATA ATT AAT GAA TAT 212 Arg His Met Ala Ser Met Ala Leu Leu Lys Asp Ile Ile Asn Glu Tyr 20 25 30 35 TTC AAT AAT ATT AAT GAT TCA AAA ATT TTG AGC CTA CAA AAC AGA AAA 260 Phe Asn Asn Ile Asn Asp Ser Lys Ile Leu Ser Leu Gln Asn Arg Lys 40 45 50 AAT ACT TTA GTG GAT ACA TCA GGA TAT AAT GCA GAA GTG AGT GAA GAA 308 Asn Thr Leu Val Asp Thr Ser Gly Tyr Asn Ala Glu Val Ser Glu Glu 55 60 65 GGC GAT GTT CAG CTT AAT CCA ATA TTT CCA TTT GAC TTT AAA TTA GGT 356 Gly Asp Val Gln Leu Asn Pro Ile Phe Pro Phe Asp Phe Lys Leu Gly 70 75 80 AGT TCA GGG GAG GAT AGA GGT AAA GTT ATA GTA ACC CAG AAT GAA AAT 404 Ser Ser Gly Glu Asp Arg Gly Lys Val Ile Val Thr Gln Asn Glu Asn 85 90 95 ATT GTA TAT AAT TCT ATG TAT GAA AGT TTT AGC ATT AGT TTT TGG ATT 452 Ile Val Tyr Asn Ser Met Tyr Glu Ser Phe Ser Ile Ser Phe Trp Ile 100 105 110 115 AGA ATA AAT AAA TGG GTA AGT AAT TTA CCT GGA TAT ACT ATA ATT GAT 500 Arg Ile Asn Lys Trp Val Ser Asn Leu Pro Gly Tyr Thr Ile Ile Asp 120 125 130 AGT GTT AAA AAT AAC TCA GGT TGG AGT ATA GGT ATT ATT AGT AAT TTT 548 Ser Val Lys Asn Asn Ser Gly Trp Ser Ile Gly Ile Ile Ser Asn Phe 135 140 145 TTA GTA TTT ACT TTA AAA CAA AAT GAA GAT AGT GAA CAA AGT ATA AAT 596 Leu Val Phe Thr Leu Lys Gln Asn Glu Asp Ser Glu Gln Ser Ile Asn 150 155 160 TTT AGT TAT GAT ATA TCA AAT AAT GCT CCT GGA TAC AAT AAA TGG TTT 644 Phe Ser Tyr Asp Ile Ser Asn Asn Ala Pro Gly Tyr Asn Lys Trp Phe 165 170 175 TTT GTA ACT GTT ACT AAC AAT ATG ATG GGA AAT ATG AAG ATT TAT ATA 692 Phe Val Thr Val Thr Asn Asn Met Met Gly Asn Met Lys Ile Tyr Ile 180 185 190 195 AAT GGA AAA TTA ATA GAT ACT ATA AAA GTT AAA GAA CTA ACT GGA ATT 740 Asn Gly Lys Leu Ile Asp Thr Ile Lys Val Lys Glu Leu Thr Gly Ile 200 205 210 AAT TTT AGC AAA ACT ATA ACA TTT GAA ATA AAT AAA ATT CCA GAT ACC 788 Asn Phe Ser Lys Thr Ile Thr Phe Glu Ile Asn Lys Ile Pro Asp Thr 215 220 225 GGT TTG ATT ACT TCA GAT TCT GAT AAC ATC AAT ATG TGG ATA AGA GAT 836 Gly Leu Ile Thr Ser Asp Ser Asp Asn Ile Asn Met Trp Ile Arg Asp 230 235 240 TTT TAT ATA TTT GCT AAA GAA TTA GAT GGT AAA GAT ATT AAT ATA TTA 884 Phe Tyr Ile Phe Ala Lys Glu Leu Asp Gly Lys Asp Ile Asn Ile Leu 245 250 255 TTT AAT AGC TTG CAA TAT ACT AAT GTT GTA AAA GAT TAT TGG GGA AAT 932 Phe Asn Ser Leu Gln Tyr Thr Asn Val Val Lys Asp Tyr Trp Gly Asn 260 265 270 275 GAT TTA AGA TAT AAT AAA GAA TAT TAT ATG GTT AAT ATA GAT TAT TTA 980 Asp Leu Arg Tyr Asn Lys Glu Tyr Tyr Met Val Asn Ile Asp Tyr Leu 280 285 290 AAT AGA TAT ATG TAT GCG AAC TCA CGA CAA ATT GTT TTT AAT ACA CGT 1028 Asn Arg Tyr Met Tyr Ala Asn Ser Arg Gln Ile Val Phe Asn Thr Arg 295 300 305 AGA AAT AAT AAT GAC TTC AAT GAA GGA TAT AAA ATT ATA ATA AAA AGA 1076 Arg Asn Asn Asn Asp Phe Asn Glu Gly Tyr Lys Ile Ile Ile Lys Arg 310 315 320 ATC AGA GGA AAT ACA AAT GAT ACT AGA GTA CGA GGA GGA GAT ATT TTA 1124 Ile Arg Gly Asn Thr Asn Asp Thr Arg Val Arg Gly Gly Asp Ile Leu 325 330 335 TAT TTT GAT ATG ACA ATT AAT AAC AAA GCA TAT AAT TTG TTT ATG AAG 1172 Tyr Phe Asp Met Thr Ile Asn Asn Lys Ala Tyr Asn Leu Phe Met Lys 340 345 350 355 AAT GAA ACT ATG TAT GCA GAT AAT CAT AGT ACT GAA GAT ATA TAT GCT 1220 Asn Glu Thr Met Tyr Ala Asp Asn His Ser Thr Glu Asp Ile Tyr Ala 360 365 370 ATA GGT TTA AGA GAA CAA ACA AAG GAT ATA AAT GAT AAT ATT ATA TTT 1268 Ile Gly Leu Arg Glu Gln Thr Lys Asp Ile Asn Asp Asn Ile Ile Phe 375 380 385 CAA ATA CAA CCA ATG AAT AAT ACT TAT TAT TAC GCA TCT CAA ATA TTT 1316 Gln Ile Gln Pro Met Asn Asn Thr Tyr Tyr Tyr Ala Ser Gln Ile Phe 390 395 400 AAA TCA AAT TTT AAT GGA GAA AAT ATT TCT GGA ATA TGT TCA ATA GGT 1364 Lys Ser Asn Phe Asn Gly Glu Asn Ile Ser Gly Ile Cys Ser Ile Gly 405 410 415 ACT TAT CGT TTT AGA CTT GGA GGT GAT TGG TAT AGA CAC AAT TAT TTG 1412 Thr Tyr Arg Phe Arg Leu Gly Gly Asp Trp Tyr Arg His Asn Tyr Leu 420 425 430 435 GTG CCT ACT GTG AAG CAA GGA AAT TAT GCT TCA TTA TTA GAA TCA ACA 1460 Val Pro Thr Val Lys Gln Gly Asn Tyr Ala Ser Leu Leu Glu Ser Thr 440 445 450 TCA ACT CAT TGG GGT TTT GTA CCT GTA AGT GAA TAAAAGCTT 1502 Ser Thr His Trp Gly Phe Val Pro Val Ser Glu 455 460

62 Met Gly His His His His His His His His His His Ser Ser Gly His 1 5 10 15 Ile Glu Gly Arg His Met Ala Ser Met Ala Leu Leu Lys Asp Ile Ile 20 25 30 Asn Glu Tyr Phe Asn Asn Ile Asn Asp Ser Lys Ile Leu Ser Leu Gln 35 40 45 Asn Arg Lys Asn Thr Leu Val Asp Thr Ser Gly Tyr Asn Ala Glu Val 50 55 60 Ser Glu Glu Gly Asp Val Gln Leu Asn Pro Ile Phe Pro Phe Asp Phe 65 70 75 80 Lys Leu Gly Ser Ser Gly Glu Asp Arg Gly Lys Val Ile Val Thr Gln 85 90 95 Asn Glu Asn Ile Val Tyr Asn Ser Met Tyr Glu Ser Phe Ser Ile Ser 100 105 110 Phe Trp Ile Arg Ile Asn Lys Trp Val Ser Asn Leu Pro Gly Tyr Thr 115 120 125 Ile Ile Asp Ser Val Lys Asn Asn Ser Gly Trp Ser Ile Gly Ile Ile 130 135 140 Ser Asn Phe Leu Val Phe Thr Leu Lys Gln Asn Glu Asp Ser Glu Gln 145 150 155 160 Ser Ile Asn Phe Ser Tyr Asp Ile Ser Asn Asn Ala Pro Gly Tyr Asn 165 170 175 Lys Trp Phe Phe Val Thr Val Thr Asn Asn Met Met Gly Asn Met Lys 180 185 190 Ile Tyr Ile Asn Gly Lys Leu Ile Asp Thr Ile Lys Val Lys Glu Leu 195 200 205 Thr Gly Ile Asn Phe Ser Lys Thr Ile Thr Phe Glu Ile Asn Lys Ile 210 215 220 Pro Asp Thr Gly Leu Ile Thr Ser Asp Ser Asp Asn Ile Asn Met Trp 225 230 235 240 Ile Arg Asp Phe Tyr Ile Phe Ala Lys Glu Leu Asp Gly Lys Asp Ile 245 250 255 Asn Ile Leu Phe Asn Ser Leu Gln Tyr Thr Asn Val Val Lys Asp Tyr 260 265 270 Trp Gly Asn Asp Leu Arg Tyr Asn Lys Glu Tyr Tyr Met Val Asn Ile 275 280 285 Asp Tyr Leu Asn Arg Tyr Met Tyr Ala Asn Ser Arg Gln Ile Val Phe 290 295 300 Asn Thr Arg Arg Asn Asn Asn Asp Phe Asn Glu Gly Tyr Lys Ile Ile 305 310 315 320 Ile Lys Arg Ile Arg Gly Asn Thr Asn Asp Thr Arg Val Arg Gly Gly 325 330 335 Asp Ile Leu Tyr Phe Asp Met Thr Ile Asn Asn Lys Ala Tyr Asn Leu 340 345 350 Phe Met Lys Asn Glu Thr Met Tyr Ala Asp Asn His Ser Thr Glu Asp 355 360 365 Ile Tyr Ala Ile Gly Leu Arg Glu Gln Thr Lys Asp Ile Asn Asp Asn 370 375 380 Ile Ile Phe Gln Ile Gln Pro Met Asn Asn Thr Tyr Tyr Tyr Ala Ser 385 390 395 400 Gln Ile Phe Lys Ser Asn Phe Asn Gly Glu Asn Ile Ser Gly Ile Cys 405 410 415 Ser Ile Gly Thr Tyr Arg Phe Arg Leu Gly Gly Asp Trp Tyr Arg His 420 425 430 Asn Tyr Leu Val Pro Thr Val Lys Gln Gly Asn Tyr Ala Ser Leu Leu 435 440 445 Glu Ser Thr Ser Thr His Trp Gly Phe Val Pro Val Ser Glu 450 455 460

63 CGCCATGGCT TTATTAAAAG ATATAATTAA TG 32

64 GCAAGCTTTT ATTCACTTAC AGGTACAAAA CC 32

65 ATG ACA TGG CCA GTA AAA GAT TTT AAT TAT AGT GAT CCT GTT AAT GAC 48 Met Thr Trp Pro Val Lys Asp Phe Asn Tyr Ser Asp Pro Val Asn Asp 1 5 10 15 AAT GAT ATA TTA TAT TTA AGA ATA CCA CAA AAT AAG TTA ATT ACT ACA 96 Asn Asp Ile Leu Tyr Leu Arg Ile Pro Gln Asn Lys Leu Ile Thr Thr 20 25 30 CCT GTA AAA GCT TTT ATG ATT ACT CAA AAT ATT TGG GTA ATA CCA GAA 144 Pro Val Lys Ala Phe Met Ile Thr Gln Asn Ile Trp Val Ile Pro Glu 35 40 45 AGA TTT TCA TCA GAT ACT AAT CCA AGT TTA AGT AAA CCG CCC AGA CCT 192 Arg Phe Ser Ser Asp Thr Asn Pro Ser Leu Ser Lys Pro Pro Arg Pro 50 55 60 ACT TCA AAG TAT CAA AGT TAT TAT GAT CCT AGT TAT TTA TCT ACT GAT 240 Thr Ser Lys Tyr Gln Ser Tyr Tyr Asp Pro Ser Tyr Leu Ser Thr Asp 65 70 75 80 GAA CAA AAA GAT ACA TTT TTA AAA GGG ATT ATA AAA TTA TTT AAA AGA 288 Glu Gln Lys Asp Thr Phe Leu Lys Gly Ile Ile Lys Leu Phe Lys Arg 85 90 95 ATT AAT GAA AGA GAT ATA GGA AAA AAA TTA ATA AAT TAT TTA GTA GTT 336 Ile Asn Glu Arg Asp Ile Gly Lys Lys Leu Ile Asn Tyr Leu Val Val 100 105 110 GGT TCA CCT TTT ATG GGA GAT TCA AGT ACG CCT GAA GAT ACA TTT GAT 384 Gly Ser Pro Phe Met Gly Asp Ser Ser Thr Pro Glu Asp Thr Phe Asp 115 120 125 TTT ACA CGT CAT ACT ACT AAT ATT GCA GTT GAA AAG TTT GAA AAT GGT 432 Phe Thr Arg His Thr Thr Asn Ile Ala Val Glu Lys Phe Glu Asn Gly 130 135 140 AGT TGG AAA GTA ACA AAT ATT ATA ACA CCA AGT GTA TTG ATA TTT GGA 480 Ser Trp Lys Val Thr Asn Ile Ile Thr Pro Ser Val Leu Ile Phe Gly 145 150 155 160 CCA CTT CCT AAT ATA TTA GAC TAT ACA GCA TCC CTT ACA TTG CAA GGA 528 Pro Leu Pro Asn Ile Leu Asp Tyr Thr Ala Ser Leu Thr Leu Gln Gly 165 170 175 CAA CAA TCA AAT CCA TCA TTT GAA GGG TTT GGA ACA TTA TCT ATA CTA 576 Gln Gln Ser Asn Pro Ser Phe Glu Gly Phe Gly Thr Leu Ser Ile Leu 180 185 190 AAA GTA GCA CCT GAA TTT TTG TTA ACA TTT AGT GAT GTA ACA TCT AAT 624 Lys Val Ala Pro Glu Phe Leu Leu Thr Phe Ser Asp Val Thr Ser Asn 195 200 205 CAA AGT TCA GCT GTA TTA GGC AAA TCT ATA TTT TGT ATG GAT CCA GTA 672 Gln Ser Ser Ala Val Leu Gly Lys Ser Ile Phe Cys Met Asp Pro Val 210 215 220 ATA GCT TTA ATG CAT GAG TTA ACA CAT TCT TTG CAT CAA TTA TAT GGA 720 Ile Ala Leu Met His Glu Leu Thr His Ser Leu His Gln Leu Tyr Gly 225 230 235 240 ATA AAT ATA CCA TCT GAT AAA AGG ATT CGT CCA CAA GTT AGC GAG GGA 768 Ile Asn Ile Pro Ser Asp Lys Arg Ile Arg Pro Gln Val Ser Glu Gly 245 250 255 TTT TTC TCT CAA GAT GGA CCC AAC GTA CAA TTT GAG GAA TTA TAT ACA 816 Phe Phe Ser Gln Asp Gly Pro Asn Val Gln Phe Glu Glu Leu Tyr Thr 260 265 270 TTT GGA GGA TTA GAT GTT GAA ATA ATA CCT CAA ATT GAA AGA TCA CAA 864 Phe Gly Gly Leu Asp Val Glu Ile Ile Pro Gln Ile Glu Arg Ser Gln 275 280 285 TTA AGA GAA AAA GCA TTA GGT CAC TAT AAA GAT ATA GCG AAA AGA CTT 912 Leu Arg Glu Lys Ala Leu Gly His Tyr Lys Asp Ile Ala Lys Arg Leu 290 295 300 AAT AAT ATT AAT AAA ACT ATT CCT TCT AGT TGG ATT AGT AAT ATA GAT 960 Asn Asn Ile Asn Lys Thr Ile Pro Ser Ser Trp Ile Ser Asn Ile Asp 305 310 315 320 AAA TAT AAA AAA ATA TTT TCT GAA AAG TAT AAT TTT GAT AAA GAT AAT 1008 Lys Tyr Lys Lys Ile Phe Ser Glu Lys Tyr Asn Phe Asp Lys Asp Asn 325 330 335 ACA GGA AAT TTT GTT GTA AAT ATT GAT AAA TTC AAT AGC TTA TAT TCA 1056 Thr Gly Asn Phe Val Val Asn Ile Asp Lys Phe Asn Ser Leu Tyr Ser 340 345 350 GAC TTG ACT AAT GTT ATG TCA GAA GTT GTT TAT TCT TCG CAA TAT AAT 1104 Asp Leu Thr Asn Val Met Ser Glu Val Val Tyr Ser Ser Gln Tyr Asn 355 360 365 GTT AAA AAC AGG ACT CAT TAT TTT TCA AGG CAT TAT CTA CCT GTA TTT 1152 Val Lys Asn Arg Thr His Tyr Phe Ser Arg His Tyr Leu Pro Val Phe 370 375 380 GCA AAT ATA TTA GAT GAT AAT ATT TAT ACT ATA AGA GAT GGT TTT AAT 1200 Ala Asn Ile Leu Asp Asp Asn Ile Tyr Thr Ile Arg Asp Gly Phe Asn 385 390 395 400 TTA ACA AAT AAA GGT TTT AAT ATA GAA AAT TCG GGT CAG AAT ATA GAA 1248 Leu Thr Asn Lys Gly Phe Asn Ile Glu Asn Ser Gly Gln Asn Ile Glu 405 410 415 AGG AAT CCT GCA CTA CAA AAG CTT AGT TCA GAA AGT GTA GTA GAT TTA 1296 Arg Asn Pro Ala Leu Gln Lys Leu Ser Ser Glu Ser Val Val Asp Leu 420 425 430 TTT ACA AAA GTA TGT TTA AGA TTA ACA AAA AAT AGT AGA GAT GAT TCA 1344 Phe Thr Lys Val Cys Leu Arg Leu Thr Lys Asn Ser Arg Asp Asp Ser 435 440 445 ACA TGT ATT AAA GTT AAA AAT AAT AGA TTA CCT TAT GTA GCT GAT AAA 1392 Thr Cys Ile Lys Val Lys Asn Asn Arg Leu Pro Tyr Val Ala Asp Lys 450 455 460 GAT AGC ATT TCA CAA GAA ATA TTT GAA AAT AAA ATT ATT ACA GAT GAG 1440 Asp Ser Ile Ser Gln Glu Ile Phe Glu Asn Lys Ile Ile Thr Asp Glu 465 470 475 480 ACT AAT GTA CAA AAT TAT TCA GAT AAT TTT TCA TTA GAT GAA TCT ATT 1488 Thr Asn Val Gln Asn Tyr Ser Asp Asn Phe Ser Leu Asp Glu Ser Ile 485 490 495 TTA GAT GGG CAA GTT CCT ATT AAT CCT GAA ATA GTA GAT CCA CTA TTA 1536 Leu Asp Gly Gln Val Pro Ile Asn Pro Glu Ile Val Asp Pro Leu Leu 500 505 510 CCC AAT GTT AAT ATG GAA CCT TTA AAT CTT CCA GGT GAA GAA ATA GTA 1584 Pro Asn Val Asn Met Glu Pro Leu Asn Leu Pro Gly Glu Glu Ile Val 515 520 525 TTT TAT GAT GAT ATT ACT AAA TAT GTT GAT TAT TTA AAT TCT TAT TAT 1632 Phe Tyr Asp Asp Ile Thr Lys Tyr Val Asp Tyr Leu Asn Ser Tyr Tyr 530 535 540 TAT TTG GAA TCT CAA AAA TTA AGT AAT AAT GTT GAA AAT ATT ACT CTT 1680 Tyr Leu Glu Ser Gln Lys Leu Ser Asn Asn Val Glu Asn Ile Thr Leu 545 550 555 560 ACA ACT TCA GTT GAA GAA GCA TTA GGT TAT AGC AAT AAG ATA TAC ACA 1728 Thr Thr Ser Val Glu Glu Ala Leu Gly Tyr Ser Asn Lys Ile Tyr Thr 565 570 575 TTT TTA CCT AGC TTA GCT GAA AAA GTG AAT AAA GGT GTT CAA GCA GGT 1776 Phe Leu Pro Ser Leu Ala Glu Lys Val Asn Lys Gly Val Gln Ala Gly 580 585 590 TTA TTC TTA AAT TGG GCG AAT GAA GTA GTT GAG GAT TTT ACT ACA AAT 1824 Leu Phe Leu Asn Trp Ala Asn Glu Val Val Glu Asp Phe Thr Thr Asn 595 600 605 ATT ATG AAG AAA GAT ACA TTG GAT AAA ATA TCA GAT GTA TCA GTA ATA 1872 Ile Met Lys Lys Asp Thr Leu Asp Lys Ile Ser Asp Val Ser Val Ile 610 615 620 ATT CCA TAT ATA GGA CCT GCC TTA AAT ATA GGA AAT TCA GCA TTA AGG 1920 Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Gly Asn Ser Ala Leu Arg 625 630 635 640 GGA AAT TTT AAG CAA GCA TTT GCA ACA GCT GGT GTA GCT TTT TTA TTA 1968 Gly Asn Phe Lys Gln Ala Phe Ala Thr Ala Gly Val Ala Phe Leu Leu 645 650 655 GAG GGA TTT CCA GAG TTT ACT ATA CCT GCA CTC GGT GTA TTT ACC TTT 2016 Glu Gly Phe Pro Glu Phe Thr Ile Pro Ala Leu Gly Val Phe Thr Phe 660 665 670 TAT AGT TCT ATT CAA GAA AGA GAG AAA ATT ATT AAA ACT ATA GAA AAT 2064 Tyr Ser Ser Ile Gln Glu Arg Glu Lys Ile Ile Lys Thr Ile Glu Asn 675 680 685 TGT TTG GAA CAA AGA GTT AAG AGA TGG AAA GAT TCA TAT CAA TGG ATG 2112 Cys Leu Glu Gln Arg Val Lys Arg Trp Lys Asp Ser Tyr Gln Trp Met 690 695 700 GTA TCA AAT TGG TTG TCA AGA ATT ACT ACT CAA TTT AAT CAT ATA AAT 2160 Val Ser Asn Trp Leu Ser Arg Ile Thr Thr Gln Phe Asn His Ile Asn 705 710 715 720 TAT CAA ATG TAT GAT TCT TTA AGT TAT CAG GCA GAT GCA ATC AAA GCT 2208 Tyr Gln Met Tyr Asp Ser Leu Ser Tyr Gln Ala Asp Ala Ile Lys Ala 725 730 735 AAA ATA GAT TTA GAA TAT AAA AAA TAC TCA GGA AGT GAT AAA GAA AAT 2256 Lys Ile Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser Asp Lys Glu Asn 740 745 750 ATA AAA AGT CAA GTT GAA AAT TTA AAA AAT AGT TTA GAT GTA AAA ATT 2304 Ile Lys Ser Gln Val Glu Asn Leu Lys Asn Ser Leu Asp Val Lys Ile 755 760 765 TCG GAA GCA ATG AAT AAT ATA AAT AAA TTT ATA CGA GAA TGT TCT GTA 2352 Ser Glu Ala Met Asn Asn Ile Asn Lys Phe Ile Arg Glu Cys Ser Val 770 775 780 ACA TAC TTA TTT AAA AAT ATG CTC CCT AAA GTA ATT GAC GAA TTA AAT 2400 Thr Tyr Leu Phe Lys Asn Met Leu Pro Lys Val Ile Asp Glu Leu Asn 785 790 795 800 AAG TTT GAT TTA AGA ACT AAA ACA GAA TTA ATT AAT CTT ATA GAT AGT 2448 Lys Phe Asp Leu Arg Thr Lys Thr Glu Leu Ile Asn Leu Ile Asp Ser 805 810 815 CAT AAT ATT ATT CTA GTT GGT GAA GTA GAT AGA TTA AAA GCA AAA GTA 2496 His Asn Ile Ile Leu Val Gly Glu Val Asp Arg Leu Lys Ala Lys Val 820 825 830 AAT GAG AGT TTT GAA AAT ACA ATG CCT TTT AAT ATT TTT TCA TAT ACT 2544 Asn Glu Ser Phe Glu Asn Thr Met Pro Phe Asn Ile Phe Ser Tyr Thr 835 840 845 AAT AAT TCT TTA TTA AAA GAT ATA ATT AAT GAA TAT TTC AAT AGT ATT 2592 Asn Asn Ser Leu Leu Lys Asp Ile Ile Asn Glu Tyr Phe Asn Ser Ile 850 855 860 AAT GAT TCA AAA ATT TTG AGC TTA CAA AAC AAA AAA AAT GCT TTA GTG 2640 Asn Asp Ser Lys Ile Leu Ser Leu Gln Asn Lys Lys Asn Ala Leu Val 865 870 875 880 GAT ACA TCA GGA TAT AAT GCA GAA GTG AGG GTA GGA GAT AAT GTT CAA 2688 Asp Thr Ser Gly Tyr Asn Ala Glu Val Arg Val Gly Asp Asn Val Gln 885 890 895 CTT AAT ACG ATA TAT ACA AAT GAC TTT AAA TTA AGT AGT TCA GGA GAT 2736 Leu Asn Thr Ile Tyr Thr Asn Asp Phe Lys Leu Ser Ser Ser Gly Asp 900 905 910 AAA ATT ATA GTA AAT TTA AAT AAT AAT ATT TTA TAT AGC GCT ATT TAT 2784 Lys Ile Ile Val Asn Leu Asn Asn Asn Ile Leu Tyr Ser Ala Ile Tyr 915 920 925 GAG AAC TCT AGT GTT AGT TTT TGG ATT AAG ATA TCT AAA GAT TTA ACT 2832 Glu Asn Ser Ser Val Ser Phe Trp Ile Lys Ile Ser Lys Asp Leu Thr 930 935 940 AAT TCT CAT AAT GAA TAT ACA ATA ATT AAC AGT ATA GAA CAA AAT TCT 2880 Asn Ser His Asn Glu Tyr Thr Ile Ile Asn Ser Ile Glu Gln Asn Ser 945 950 955 960 GGG TGG AAA TTA TGT ATT AGG AAT GGC AAT ATA GAA TGG ATT TTA CAA 2928 Gly Trp Lys Leu Cys Ile Arg Asn Gly Asn Ile Glu Trp Ile Leu Gln 965 970 975 GAT GTT AAT AGA AAG TAT AAA AGT TTA ATT TTT GAT TAT AGT GAA TCA 2976 Asp Val Asn Arg Lys Tyr Lys Ser Leu Ile Phe Asp Tyr Ser Glu Ser 980 985 990 TTA AGT CAT ACA GGA TAT ACA AAT AAA TGG TTT TTT GTT ACT ATA ACT 3024 Leu Ser His Thr Gly Tyr Thr Asn Lys Trp Phe Phe Val Thr Ile Thr 995 1000 1005 AAT AAT ATA ATG GGG TAT ATG AAA CTT TAT ATA AAT GGA GAA TTA AAG 3072 Asn Asn Ile Met Gly Tyr Met Lys Leu Tyr Ile Asn Gly Glu Leu Lys 1010 1015 1020 CAG AGT CAA AAA ATT GAA GAT TTA GAT GAG GTT AAG TTA GAT AAA ACC 3120 Gln Ser Gln Lys Ile Glu Asp Leu Asp Glu Val Lys Leu Asp Lys Thr 1025 1030 1035 1040 ATA GTA TTT GGA ATA GAT GAG AAT ATA GAT GAG AAT CAG ATG CTT TGG 3168 Ile Val Phe Gly Ile Asp Glu Asn Ile Asp Glu Asn Gln Met Leu Trp 1045 1050 1055 ATT AGA GAT TTT AAT ATT TTT TCT AAA GAA TTA AGT AAT GAA GAT ATT 3216 Ile Arg Asp Phe Asn Ile Phe Ser Lys Glu Leu Ser Asn Glu Asp Ile 1060 1065 1070 AAT ATT GTA TAT GAG GGA CAA ATA TTA AGA AAT GTT ATT AAA GAT TAT 3264 Asn Ile Val Tyr Glu Gly Gln Ile Leu Arg Asn Val Ile Lys Asp Tyr 1075 1080 1085 TGG GGA AAT CCT TTG AAG TTT GAT ACA GAA TAT TAT ATT ATT AAT GAT 3312 Trp Gly Asn Pro Leu Lys Phe Asp Thr Glu Tyr Tyr Ile Ile Asn Asp 1090 1095 1100 AAT TAT ATA GAT AGG TAT ATT GCA CCT GAA AGT AAT GTA CTT GTA CTT 3360 Asn Tyr Ile Asp Arg Tyr Ile Ala Pro Glu Ser Asn Val Leu Val Leu 1105 1110 1115 1120 GTT CGG TAT CCA GAT AGA TCT AAA TTA TAT ACT GGA AAT CCT ATT ACT 3408 Val Arg Tyr Pro Asp Arg Ser Lys Leu Tyr Thr Gly Asn Pro Ile Thr 1125 1130 1135 ATT AAA TCA GTA TCT GAT AAG AAT CCT TAT AGT AGA ATT TTA AAT GGA 3456 Ile Lys Ser Val Ser Asp Lys Asn Pro Tyr Ser Arg Ile Leu Asn Gly 1140 1145 1150 GAT AAT ATA ATT CTT CAT ATG TTA TAT AAT AGT AGG AAA TAT ATG ATA 3504 Asp Asn Ile Ile Leu His Met Leu Tyr Asn Ser Arg Lys Tyr Met Ile 1155 1160 1165 ATA AGA GAT ACT GAT ACA ATA TAT GCA ACA CAA GGA GGA GAG TGT TCA 3552 Ile Arg Asp Thr Asp Thr Ile Tyr Ala Thr Gln Gly Gly Glu Cys Ser 1170 1175 1180 CAA AAT TGT GTA TAT GCA TTA AAA TTA CAG AGT AAT TTA GGT AAT TAT 3600 Gln Asn Cys Val Tyr Ala Leu Lys Leu Gln Ser Asn Leu Gly Asn Tyr 1185 1190 1195 1200 GGT ATA GGT ATA TTT AGT ATA AAA AAT ATT GTA TCT AAA AAT AAA TAT 3648 Gly Ile Gly Ile Phe Ser Ile Lys Asn Ile Val Ser Lys Asn Lys Tyr 1205 1210 1215 TGT AGT CAA ATT TTC TCT AGT TTT AGG GAA AAT ACA ATG CTT CTA GCA 3696 Cys Ser Gln Ile Phe Ser Ser Phe Arg Glu Asn Thr Met Leu Leu Ala 1220 1225 1230 GAT ATA TAT AAA CCT TGG AGA TTT TCT TTT AAA AAT GCA TAC ACG CCA 3744 Asp Ile Tyr Lys Pro Trp Arg Phe Ser Phe Lys Asn Ala Tyr Thr Pro 1235 1240 1245 GTT GCA GTA ACT AAT TAT GAA ACA AAA CTA TTA TCA ACT TCA TCT TTT 3792 Val Ala Val Thr Asn Tyr Glu Thr Lys Leu Leu Ser Thr Ser Ser Phe 1250 1255 1260 TGG AAA TTT ATT TCT AGG GAT CCA GGA TGG GTA GAG TAA 3831 Trp Lys Phe Ile Ser Arg Asp Pro Gly Trp Val Glu 1265 1270 1275

66 Met Thr Trp Pro Val Lys Asp Phe Asn Tyr Ser Asp Pro Val Asn Asp 1 5 10 15 Asn Asp Ile Leu Tyr Leu Arg Ile Pro Gln Asn Lys Leu Ile Thr Thr 20 25 30 Pro Val Lys Ala Phe Met Ile Thr Gln Asn Ile Trp Val Ile Pro Glu 35 40 45 Arg Phe Ser Ser Asp Thr Asn Pro Ser Leu Ser Lys Pro Pro Arg Pro 50 55 60 Thr Ser Lys Tyr Gln Ser Tyr Tyr Asp Pro Ser Tyr Leu Ser Thr Asp 65 70 75 80 Glu Gln Lys Asp Thr Phe Leu Lys Gly Ile Ile Lys Leu Phe Lys Arg 85 90 95 Ile Asn Glu Arg Asp Ile Gly Lys Lys Leu Ile Asn Tyr Leu Val Val 100 105 110 Gly Ser Pro Phe Met Gly Asp Ser Ser Thr Pro Glu Asp Thr Phe Asp 115 120 125 Phe Thr Arg His Thr Thr Asn Ile Ala Val Glu Lys Phe Glu Asn Gly 130 135 140 Ser Trp Lys Val Thr Asn Ile Ile Thr Pro Ser Val Leu Ile Phe Gly 145 150 155 160 Pro Leu Pro Asn Ile Leu Asp Tyr Thr Ala Ser Leu Thr Leu Gln Gly 165 170 175 Gln Gln Ser Asn Pro Ser Phe Glu Gly Phe Gly Thr Leu Ser Ile Leu 180 185 190 Lys Val Ala Pro Glu Phe Leu Leu Thr Phe Ser Asp Val Thr Ser Asn 195 200 205 Gln Ser Ser Ala Val Leu Gly Lys Ser Ile Phe Cys Met Asp Pro Val 210 215 220 Ile Ala Leu Met His Glu Leu Thr His Ser Leu His Gln Leu Tyr Gly 225 230 235 240 Ile Asn Ile Pro Ser Asp Lys Arg Ile Arg Pro Gln Val Ser Glu Gly 245 250 255 Phe Phe Ser Gln Asp Gly Pro Asn Val Gln Phe Glu Glu Leu Tyr Thr 260 265 270 Phe Gly Gly Leu Asp Val Glu Ile Ile Pro Gln Ile Glu Arg Ser Gln 275 280 285 Leu Arg Glu Lys Ala Leu Gly His Tyr Lys Asp Ile Ala Lys Arg Leu 290 295 300 Asn Asn Ile Asn Lys Thr Ile Pro Ser Ser Trp Ile Ser Asn Ile Asp 305 310 315 320 Lys Tyr Lys Lys Ile Phe Ser Glu Lys Tyr Asn Phe Asp Lys Asp Asn 325 330 335 Thr Gly Asn Phe Val Val Asn Ile Asp Lys Phe Asn Ser Leu Tyr Ser 340 345 350 Asp Leu Thr Asn Val Met Ser Glu Val Val Tyr Ser Ser Gln Tyr Asn 355 360 365 Val Lys Asn Arg Thr His Tyr Phe Ser Arg His Tyr Leu Pro Val Phe 370 375 380 Ala Asn Ile Leu Asp Asp Asn Ile Tyr Thr Ile Arg Asp Gly Phe Asn 385 390 395 400 Leu Thr Asn Lys Gly Phe Asn Ile Glu Asn Ser Gly Gln Asn Ile Glu 405 410 415 Arg Asn Pro Ala Leu Gln Lys Leu Ser Ser Glu Ser Val Val Asp Leu 420 425 430 Phe Thr Lys Val Cys Leu Arg Leu Thr Lys Asn Ser Arg Asp Asp Ser 435 440 445 Thr Cys Ile Lys Val Lys Asn Asn Arg Leu Pro Tyr Val Ala Asp Lys 450 455 460 Asp Ser Ile Ser Gln Glu Ile Phe Glu Asn Lys Ile Ile Thr Asp Glu 465 470 475 480 Thr Asn Val Gln Asn Tyr Ser Asp Asn Phe Ser Leu Asp Glu Ser Ile 485 490 495 Leu Asp Gly Gln Val Pro Ile Asn Pro Glu Ile Val Asp Pro Leu Leu 500 505 510 Pro Asn Val Asn Met Glu Pro Leu Asn Leu Pro Gly Glu Glu Ile Val 515 520 525 Phe Tyr Asp Asp Ile Thr Lys Tyr Val Asp Tyr Leu Asn Ser Tyr Tyr 530 535 540 Tyr Leu Glu Ser Gln Lys Leu Ser Asn Asn Val Glu Asn Ile Thr Leu 545 550 555 560 Thr Thr Ser Val Glu Glu Ala Leu Gly Tyr Ser Asn Lys Ile Tyr Thr 565 570 575 Phe Leu Pro Ser Leu Ala Glu Lys Val Asn Lys Gly Val Gln Ala Gly 580 585 590 Leu Phe Leu Asn Trp Ala Asn Glu Val Val Glu Asp Phe Thr Thr Asn 595 600 605 Ile Met Lys Lys Asp Thr Leu Asp Lys Ile Ser Asp Val Ser Val Ile 610 615 620 Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Gly Asn Ser Ala Leu Arg 625 630 635 640 Gly Asn Phe Lys Gln Ala Phe Ala Thr Ala Gly Val Ala Phe Leu Leu 645 650 655 Glu Gly Phe Pro Glu Phe Thr Ile Pro Ala Leu Gly Val Phe Thr Phe 660 665 670 Tyr Ser Ser Ile Gln Glu Arg Glu Lys Ile Ile Lys Thr Ile Glu Asn 675 680 685 Cys Leu Glu Gln Arg Val Lys Arg Trp Lys Asp Ser Tyr Gln Trp Met 690 695 700 Val Ser Asn Trp Leu Ser Arg Ile Thr Thr Gln Phe Asn His Ile Asn 705 710 715 720 Tyr Gln Met Tyr Asp Ser Leu Ser Tyr Gln Ala Asp Ala Ile Lys Ala 725 730 735 Lys Ile Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser Asp Lys Glu Asn 740 745 750 Ile Lys Ser Gln Val Glu Asn Leu Lys Asn Ser Leu Asp Val Lys Ile 755 760 765 Ser Glu Ala Met Asn Asn Ile Asn Lys Phe Ile Arg Glu Cys Ser Val 770 775 780 Thr Tyr Leu Phe Lys Asn Met Leu Pro Lys Val Ile Asp Glu Leu Asn 785 790 795 800 Lys Phe Asp Leu Arg Thr Lys Thr Glu Leu Ile Asn Leu Ile Asp Ser 805 810 815 His Asn Ile Ile Leu Val Gly Glu Val Asp Arg Leu Lys Ala Lys Val 820 825 830 Asn Glu Ser Phe Glu Asn Thr Met Pro Phe Asn Ile Phe Ser Tyr Thr 835 840 845 Asn Asn Ser Leu Leu Lys Asp Ile Ile Asn Glu Tyr Phe Asn Ser Ile 850 855 860 Asn Asp Ser Lys Ile Leu Ser Leu Gln Asn Lys Lys Asn Ala Leu Val 865 870 875 880 Asp Thr Ser Gly Tyr Asn Ala Glu Val Arg Val Gly Asp Asn Val Gln 885 890 895 Leu Asn Thr Ile Tyr Thr Asn Asp Phe Lys Leu Ser Ser Ser Gly Asp 900 905 910 Lys Ile Ile Val Asn Leu Asn Asn Asn Ile Leu Tyr Ser Ala Ile Tyr 915 920 925 Glu Asn Ser Ser Val Ser Phe Trp Ile Lys Ile Ser Lys Asp Leu Thr 930 935 940 Asn Ser His Asn Glu Tyr Thr Ile Ile Asn Ser Ile Glu Gln Asn Ser 945 950 955 960 Gly Trp Lys Leu Cys Ile Arg Asn Gly Asn Ile Glu Trp Ile Leu Gln 965 970 975 Asp Val Asn Arg Lys Tyr Lys Ser Leu Ile Phe Asp Tyr Ser Glu Ser 980 985 990 Leu Ser His Thr Gly Tyr Thr Asn Lys Trp Phe Phe Val Thr Ile Thr 995 1000 1005 Asn Asn Ile Met Gly Tyr Met Lys Leu Tyr Ile Asn Gly Glu Leu Lys 1010 1015 1020 Gln Ser Gln Lys Ile Glu Asp Leu Asp Glu Val Lys Leu Asp Lys Thr 1025 1030 1035 1040 Ile Val Phe Gly Ile Asp Glu Asn Ile Asp Glu Asn Gln Met Leu Trp 1045 1050 1055 Ile Arg Asp Phe Asn Ile Phe Ser Lys Glu Leu Ser Asn Glu Asp Ile 1060 1065 1070 Asn Ile Val Tyr Glu Gly Gln Ile Leu Arg Asn Val Ile Lys Asp Tyr 1075 1080 1085 Trp Gly Asn Pro Leu Lys Phe Asp Thr Glu Tyr Tyr Ile Ile Asn Asp 1090 1095 1100 Asn Tyr Ile Asp Arg Tyr Ile Ala Pro Glu Ser Asn Val Leu Val Leu 1105 1110 1115 1120 Val Arg Tyr Pro Asp Arg Ser Lys Leu Tyr Thr Gly Asn Pro Ile Thr 1125 1130 1135 Ile Lys Ser Val Ser Asp Lys Asn Pro Tyr Ser Arg Ile Leu Asn Gly 1140 1145 1150 Asp Asn Ile Ile Leu His Met Leu Tyr Asn Ser Arg Lys Tyr Met Ile 1155 1160 1165 Ile Arg Asp Thr Asp Thr Ile Tyr Ala Thr Gln Gly Gly Glu Cys Ser 1170 1175 1180 Gln Asn Cys Val Tyr Ala Leu Lys Leu Gln Ser Asn Leu Gly Asn Tyr 1185 1190 1195 1200 Gly Ile Gly Ile Phe Ser Ile Lys Asn Ile Val Ser Lys Asn Lys Tyr 1205 1210 1215 Cys Ser Gln Ile Phe Ser Ser Phe Arg Glu Asn Thr Met Leu Leu Ala 1220 1225 1230 Asp Ile Tyr Lys Pro Trp Arg Phe Ser Phe Lys Asn Ala Tyr Thr Pro 1235 1240 1245 Val Ala Val Thr Asn Tyr Glu Thr Lys Leu Leu Ser Thr Ser Ser Phe 1250 1255 1260 Trp Lys Phe Ile Ser Arg Asp Pro Gly Trp Val Glu 1265 1270 1275

67 AGATCTCGAT CCCGCGAAAT TAATACGACT CACTATAGGG GAATTGTGAG CGGATAACAA 60 TTCCCCTCTA GAAATAATTT TGTTTAACTT TAAGAAGGAG ATATACC ATG GGC CAT 116 Met Gly His 1 CAT CAT CAT CAT CAT CAT CAT CAT CAC AGC AGC GGC CAT ATC GAA GGT 164 His His His His His His His His His Ser Ser Gly His Ile GluGly 5 10 15 CGT CAT ATG GCT AGC ATG GCT TTA TTA AAA GAT ATA ATT AAT GAA TAT 212 Arg His Met Ala Ser Met Ala Leu Leu Lys Asp Ile Ile Asn Glu Tyr 20 25 30 35 TTC AAT AGT ATT AAT GAT TCA AAA ATT TTG AGC TTA CAA AAC AAA AAA 260 Phe Asn Ser Ile Asn Asp Ser Lys Ile Leu Ser Leu Gln Asn Lys Lys 40 45 50 AAT GCT TTA GTG GAT ACA TCA GGA TAT AAT GCA GAA GTG AGG GTA GGA 308 Asn Ala Leu Val Asp Thr Ser Gly Tyr Asn Ala Glu Val Arg Val Gly 55 60 65 GAT AAT GTT CAA CTT AAT ACG ATA TAT ACA AAT GAC TTT AAA TTA AGT 356 Asp Asn Val Gln Leu Asn Thr Ile Tyr Thr Asn Asp Phe Lys Leu Ser 70 75 80 AGT TCA GGA GAT AAA ATT ATA GTA AAT TTA AAT AAT AAT ATT TTA TAT 404 Ser Ser Gly Asp Lys Ile Ile Val Asn Leu Asn Asn Asn Ile Leu Tyr 85 90 95 AGC GCT ATT TAT GAG AAC TCT AGT GTT AGT TTT TGG ATT AAG ATA TCT 452 Ser Ala Ile Tyr Glu Asn Ser Ser Val Ser Phe Trp Ile Lys Ile Ser 100 105 110 115 AAA GAT TTA ACT AAT TCT CAT AAT GAA TAT ACA ATA ATT AAC AGT ATA 500 Lys Asp Leu Thr Asn Ser His Asn Glu Tyr Thr Ile Ile Asn Ser Ile 120 125 130 GAA CAA AAT TCT GGG TGG AAA TTA TGT ATT AGG AAT GGC AAT ATA GAA 548 Glu Gln Asn Ser Gly Trp Lys Leu Cys Ile Arg Asn Gly Asn Ile Glu 135 140 145 TGG ATT TTA CAA GAT GTT AAT AGA AAG TAT AAA AGT TTA ATT TTT GAT 596 Trp Ile Leu Gln Asp Val Asn Arg Lys Tyr Lys Ser Leu Ile Phe Asp 150 155 160 TAT AGT GAA TCA TTA AGT CAT ACA GGA TAT ACA AAT AAA TGG TTT TTT 644 Tyr Ser Glu Ser Leu Ser His Thr Gly Tyr Thr Asn Lys Trp Phe Phe 165 170 175 GTT ACT ATA ACT AAT AAT ATA ATG GGG TAT ATG AAA CTT TAT ATA AAT 692 Val Thr Ile Thr Asn Asn Ile Met Gly Tyr Met Lys Leu Tyr Ile Asn 180 185 190 195 GGA GAA TTA AAG CAG AGT CAA AAA ATT GAA GAT TTA GAT GAG GTT AAG 740 Gly Glu Leu Lys Gln Ser Gln Lys Ile Glu Asp Leu Asp Glu Val Lys 200 205 210 TTA GAT AAA ACC ATA GTA TTT GGA ATA GAT GAG AAT ATA GAT GAG AAT 788 Leu Asp Lys Thr Ile Val Phe Gly Ile Asp Glu Asn Ile Asp Glu Asn 215 220 225 CAG ATG CTT TGG ATT AGA GAT TTT AAT ATT TTT TCT AAA GAA TTA AGT 836 Gln Met Leu Trp Ile Arg Asp Phe Asn Ile Phe Ser Lys Glu Leu Ser 230 235 240 AAT GAA GAT ATT AAT ATT GTA TAT GAG GGA CAA ATA TTA AGA AAT GTT 884 Asn Glu Asp Ile Asn Ile Val Tyr Glu Gly Gln Ile Leu Arg Asn Val 245 250 255 ATT AAA GAT TAT TGG GGA AAT CCT TTG AAG TTT GAT ACA GAA TAT TAT 932 Ile Lys Asp Tyr Trp Gly Asn Pro Leu Lys Phe Asp Thr Glu Tyr Tyr 260 265 270 275 ATT ATT AAT GAT AAT TAT ATA GAT AGG TAT ATT GCA CCT GAA AGT AAT 980 Ile Ile Asn Asp Asn Tyr Ile Asp Arg Tyr Ile Ala Pro Glu Ser Asn 280 285 290 GTA CTT GTA CTT GTT CGG TAT CCA GAT AGA TCT AAA TTA TAT ACT GGA 1028 Val Leu Val Leu Val Arg Tyr Pro Asp Arg Ser Lys Leu Tyr Thr Gly 295 300 305 AAT CCT ATT ACT ATT AAA TCA GTA TCT GAT AAG AAT CCT TAT AGT AGA 1076 Asn Pro Ile Thr Ile Lys Ser Val Ser Asp Lys Asn Pro Tyr Ser Arg 310 315 320 ATT TTA AAT GGA GAT AAT ATA ATT CTT CAT ATG TTA TAT AAT AGT AGG 1124 Ile Leu Asn Gly Asp Asn Ile Ile Leu His Met Leu Tyr Asn Ser Arg 325 330 335 AAA TAT ATG ATA ATA AGA GAT ACT GAT ACA ATA TAT GCA ACA CAA GGA 1172 Lys Tyr Met Ile Ile Arg Asp Thr Asp Thr Ile Tyr Ala Thr Gln Gly 340 345 350 355 GGA GAG TGT TCA CAA AAT TGT GTA TAT GCA TTA AAA TTA CAG AGT AAT 1220 Gly Glu Cys Ser Gln Asn Cys Val Tyr Ala Leu Lys Leu Gln Ser Asn 360 365 370 TTA GGT AAT TAT GGT ATA GGT ATA TTT AGT ATA AAA AAT ATT GTA TCT 1268 Leu Gly Asn Tyr Gly Ile Gly Ile Phe Ser Ile Lys Asn Ile Val Ser 375 380 385 AAA AAT AAA TAT TGT AGT CAA ATT TTC TCT AGT TTT AGG GAA AAT ACA 1316 Lys Asn Lys Tyr Cys Ser Gln Ile Phe Ser Ser Phe Arg Glu Asn Thr 390 395 400 ATG CTT CTA GCA GAT ATA TAT AAA CCT TGG AGA TTT TCT TTT AAA AAT 1364 Met Leu Leu Ala Asp Ile Tyr Lys Pro Trp Arg Phe Ser Phe Lys Asn 405 410 415 GCA TAC ACG CCA GTT GCA GTA ACT AAT TAT GAA ACA AAA CTA TTA TCA 1412 Ala Tyr Thr Pro Val Ala Val Thr Asn Tyr Glu Thr Lys Leu Leu Ser 420 425 430 435 ACT TCA TCT TTT TGG AAA TTT ATT TCT AGG GAT CCA GGA TGG GTA GAG 1460 Thr Ser Ser Phe Trp Lys Phe Ile Ser Arg Asp Pro Gly Trp Val Glu 440 445 450 TAAAAGCTT 1469

68 Met Gly His His His His His His His His His His Ser Ser Gly His 1 5 10 15 Ile Glu Gly Arg His Met Ala Ser Met Ala Leu Leu Lys Asp Ile Ile 20 25 30 Asn Glu Tyr Phe Asn Ser Ile Asn Asp Ser Lys Ile Leu Ser Leu Gln 35 40 45 Asn Lys Lys Asn Ala Leu Val Asp Thr Ser Gly Tyr Asn Ala Glu Val 50 55 60 Arg Val Gly Asp Asn Val Gln Leu Asn Thr Ile Tyr Thr Asn Asp Phe 65 70 75 80 Lys Leu Ser Ser Ser Gly Asp Lys Ile Ile Val Asn Leu Asn Asn Asn 85 90 95 Ile Leu Tyr Ser Ala Ile Tyr Glu Asn Ser Ser Val Ser Phe Trp Ile 100 105 110 Lys Ile Ser Lys Asp Leu Thr Asn Ser His Asn Glu Tyr Thr Ile Ile 115 120 125 Asn Ser Ile Glu Gln Asn Ser Gly Trp Lys Leu Cys Ile Arg Asn Gly 130 135 140 Asn Ile Glu Trp Ile Leu Gln Asp Val Asn Arg Lys Tyr Lys Ser Leu 145 150 155 160 Ile Phe Asp Tyr Ser Glu Ser Leu Ser His Thr Gly Tyr Thr Asn Lys 165 170 175 Trp Phe Phe Val Thr Ile Thr Asn Asn Ile Met Gly Tyr Met Lys Leu 180 185 190 Tyr Ile Asn Gly Glu Leu Lys Gln Ser Gln Lys Ile Glu Asp Leu Asp 195 200 205 Glu Val Lys Leu Asp Lys Thr Ile Val Phe Gly Ile Asp Glu Asn Ile 210 215 220 Asp Glu Asn Gln Met Leu Trp Ile Arg Asp Phe Asn Ile Phe Ser Lys 225 230 235 240 Glu Leu Ser Asn Glu Asp Ile Asn Ile Val Tyr Glu Gly Gln Ile Leu 245 250 255 Arg Asn Val Ile Lys Asp Tyr Trp Gly Asn Pro Leu Lys Phe Asp Thr 260 265 270 Glu Tyr Tyr Ile Ile Asn Asp Asn Tyr Ile Asp Arg Tyr Ile Ala Pro 275 280 285 Glu Ser Asn Val Leu Val Leu Val Arg Tyr Pro Asp Arg Ser Lys Leu 290 295 300 Tyr Thr Gly Asn Pro Ile Thr Ile Lys Ser Val Ser Asp Lys Asn Pro 305 310 315 320 Tyr Ser Arg Ile Leu Asn Gly Asp Asn Ile Ile Leu His Met Leu Tyr 325 330 335 Asn Ser Arg Lys Tyr Met Ile Ile Arg Asp Thr Asp Thr Ile Tyr Ala 340 345 350 Thr Gln Gly Gly Glu Cys Ser Gln Asn Cys Val Tyr Ala Leu Lys Leu 355 360 365 Gln Ser Asn Leu Gly Asn Tyr Gly Ile Gly Ile Phe Ser Ile Lys Asn 370 375 380 Ile Val Ser Lys Asn Lys Tyr Cys Ser Gln Ile Phe Ser Ser Phe Arg 385 390 395 400 Glu Asn Thr Met Leu Leu Ala Asp Ile Tyr Lys Pro Trp Arg Phe Ser 405 410 415 Phe Lys Asn Ala Tyr Thr Pro Val Ala Val Thr Asn Tyr Glu Thr Lys 420 425 430 Leu Leu Ser Thr Ser Ser Phe Trp Lys Phe Ile Ser Arg Asp Pro Gly 435 440 445 Trp Val Glu 450

69 GCAAGCTTTT ACTCTACCCA TCCTGGATCC CT 32

70 ATG CCA GTT GCA ATA AAT AGT TTT AAT TAT AAT GAC CCT GTT AAT GAT 48 Met Pro Val Ala Ile Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asp 1 5 10 15 GAT ACA ATT TTA TAC ATG CAG ATA CCA TAT GAA GAA AAA AGT AAA AAA 96 Asp Thr Ile Leu Tyr Met Gln Ile Pro Tyr Glu Glu Lys Ser Lys Lys 20 25 30 TAT TAT AAA GCT TTT GAG ATT ATG CGT AAT GTT TGG ATA ATT CCT GAG 144 Tyr Tyr Lys Ala Phe Glu Ile Met Arg Asn Val Trp Ile Ile Pro Glu 35 40 45 AGA AAT ACA ATA GGA ACG AAT CCT AGT GAT TTT GAT CCA CCG GCT TCA 192 Arg Asn Thr Ile Gly Thr Asn Pro Ser Asp Phe Asp Pro Pro Ala Ser 50 55 60 TTA AAG AAC GGA AGC AGT GCT TAT TAT GAT CCT AAT TAT TTA ACC ACT 240 Leu Lys Asn Gly Ser Ser Ala Tyr Tyr Asp Pro Asn Tyr Leu Thr Thr 65 70 75 80 GAT GCT GAA AAA GAT AGA TAT TTA AAA ACA ACG ATA AAA TTA TTT AAG 288 Asp Ala Glu Lys Asp Arg Tyr Leu Lys Thr Thr Ile Lys Leu Phe Lys 85 90 95 AGA ATT AAT AGT AAT CCT GCA GGG AAA GTT TTG TTA CAA GAA ATA TCA 336 Arg Ile Asn Ser Asn Pro Ala Gly Lys Val Leu Leu Gln Glu Ile Ser 100 105 110 TAT GCT AAA CCA TAT TTA GGA AAT GAC CAC ACG CCA ATT GAT GAA TTC 384 Tyr Ala Lys Pro Tyr Leu Gly Asn Asp His Thr Pro Ile Asp Glu Phe 115 120 125 TCT CCA GTT ACT AGA ACT ACA AGT GTT AAT ATA AAA TTA TCA ACT AAT 432 Ser Pro Val Thr Arg Thr Thr Ser Val Asn Ile Lys Leu Ser Thr Asn 130 135 140 GTT GAA AGT TCA ATG TTA TTG AAT CTT CTT GTA TTG GGA GCA GGA CCT 480 Val Glu Ser Ser Met Leu Leu Asn Leu Leu Val Leu Gly Ala Gly Pro 145 150 155 160 GAT ATA TTT GAA AGT TGT TGT TAC CCC GTT AGA AAA CTA ATA GAT CCA 528 Asp Ile Phe Glu Ser Cys Cys Tyr Pro Val Arg Lys Leu Ile Asp Pro 165 170 175 GAT GTA GTT TAT GAT CCA AGT AAT TAT GGT TTT GGA TCA ATT AAT ATC 576 Asp Val Val Tyr Asp Pro Ser Asn Tyr Gly Phe Gly Ser Ile Asn Ile 180 185 190 GTG ACA TTT TCA CCT GAG TAT GAA TAT ACT TTT AAT GAT ATT AGT GGA 624 Val Thr Phe Ser Pro Glu Tyr Glu Tyr Thr Phe Asn Asp Ile Ser Gly 195 200 205 GGG CAT AAT AGT AGT ACA GAA TCA TTT ATT GCA GAT CCT GCA ATT TCA 672 Gly His Asn Ser Ser Thr Glu Ser Phe Ile Ala Asp Pro Ala Ile Ser 210 215 220 CTA GCT CAT GAA TTG ATA CAT GCA CTG CAT GGA TTA TAC GGG GCT AGG 720 Leu Ala His Glu Leu Ile His Ala Leu His Gly Leu Tyr Gly Ala Arg 225 230 235 240 GGA GTT ACT TAT GAA GAG ACT ATA GAA GTA AAG CAA GCA CCT CTT ATG 768 Gly Val Thr Tyr Glu Glu Thr Ile Glu Val Lys Gln Ala Pro Leu Met 245 250 255 ATA GCC GAA AAA CCC ATA AGG CTA GAA GAA TTT TTA ACC TTT GGA GGT 816 Ile Ala Glu Lys Pro Ile Arg Leu Glu Glu Phe Leu Thr Phe Gly Gly 260 265 270 CAG GAT TTA AAT ATT ATT ACT AGT GCT ATG AAG GAA AAA ATA TAT AAC 864 Gln Asp Leu Asn Ile Ile Thr Ser Ala Met Lys Glu Lys Ile Tyr Asn 275 280 285 AAT CTT TTA GCT AAC TAT GAA AAA ATA GCT ACT AGA CTT AGT GAA GTT 912 Asn Leu Leu Ala Asn Tyr Glu Lys Ile Ala Thr Arg Leu Ser Glu Val 290 295 300 AAT AGT GCT CCT CCT GAA TAT GAT ATT AAT GAA TAT AAA GAT TAT TTT 960 Asn Ser Ala Pro Pro Glu Tyr Asp Ile Asn Glu Tyr Lys Asp Tyr Phe 305 310 315 320 CAA TGG AAG TAT GGG CTA GAT AAA AAT GCT GAT GGA AGT TAT ACT GTA 1008 Gln Trp Lys Tyr Gly Leu Asp Lys Asn Ala Asp Gly Ser Tyr Thr Val 325 330 335 AAT GAA AAT AAA TTT AAT GAA ATT TAT AAA AAA TTA TAT AGT TTT ACA 1056 Asn Glu Asn Lys Phe Asn Glu Ile Tyr Lys Lys Leu Tyr Ser Phe Thr 340 345 350 GAG AGT GAC TTA GCA AAT AAA TTT AAA GTA AAA TGT AGA AAT ACT TAT 1104 Glu Ser Asp Leu Ala Asn Lys Phe Lys Val Lys Cys Arg Asn Thr Tyr 355 360 365 TTT ATT AAA TAT GAA TTT TTA AAA GTT CCA AAT TTG TTA GAT GAT GAT 1152 Phe Ile Lys Tyr Glu Phe Leu Lys Val Pro Asn Leu Leu Asp Asp Asp 370 375 380 ATT TAT ACT GTA TCA GAG GGG TTT AAT ATA GGT AAT TTA GCA GTA AAC 1200 Ile Tyr Thr Val Ser Glu Gly Phe Asn Ile Gly Asn Leu Ala Val Asn 385 390 395 400 AAT CGC GGA CAA AGT ATA AAG TTA AAT CCT AAA ATT ATT GAT TCC ATT 1248 Asn Arg Gly Gln Ser Ile Lys Leu Asn Pro Lys Ile Ile Asp Ser Ile 405 410 415 CCA GAT AAA GGT CTA GTA GAA AAG ATC GTT AAA TTT TGT AAG AGC GTT 1296 Pro Asp Lys Gly Leu Val Glu Lys Ile Val Lys Phe Cys Lys Ser Val 420 425 430 ATT CCT AGA AAA GGT ACA AAG GCG CCA CCG CGA CTA TGC ATT AGA GTA 1344 Ile Pro Arg Lys Gly Thr Lys Ala Pro Pro Arg Leu Cys Ile Arg Val 435 440 445 AAT AAT AGT GAG TTA TTT TTT GTA GCT TCA GAA AGT AGC TAT AAT GAA 1392 Asn Asn Ser Glu Leu Phe Phe Val Ala Ser Glu Ser Ser Tyr Asn Glu 450 455 460 AAT GAT ATT AAT ACA CCT AAA GAA ATT GAC GAT ACA ACA AAT CTA AAT 1440 Asn Asp Ile Asn Thr Pro Lys Glu Ile Asp Asp Thr Thr Asn Leu Asn 465 470 475 480 AAT AAT TAT AGA AAT AAT TTA GAT GAA GTT ATT TTA GAT TAT AAT AGT 1488 Asn Asn Tyr Arg Asn Asn Leu Asp Glu Val Ile Leu Asp Tyr Asn Ser 485 490 495 CAG ACA ATA CCT CAA ATA TCA AAT CGA ACA TTA AAT ACA CTT GTA CAA 1536 Gln Thr Ile Pro Gln Ile Ser Asn Arg Thr Leu Asn Thr Leu Val Gln 500 505 510 GAC AAT AGT TAT GTG CCA AGA TAT GAT TCT AAT GGA ACA AGT GAA ATA 1584 Asp Asn Ser Tyr Val Pro Arg Tyr Asp Ser Asn Gly Thr Ser Glu Ile 515 520 525 GAG GAA TAT GAT GTT GTT GAC TTT AAT GTA TTT TTC TAT TTA CAT GCA 1632 Glu Glu Tyr Asp Val Val Asp Phe Asn Val Phe Phe Tyr Leu His Ala 530 535 540 CAA AAA GTG CCA GAA GGT GAA ACC AAT ATA AGT TTA ACT TCT TCA ATT 1680 Gln Lys Val Pro Glu Gly Glu Thr Asn Ile Ser Leu Thr Ser Ser Ile 545 550 555 560 GAT ACA GCA TTA TTA GAA GAA TCC AAA GAT ATA TTT TTT TCT TCA GAG 1728 Asp Thr Ala Leu Leu Glu Glu Ser Lys Asp Ile Phe Phe Ser Ser Glu 565 570 575 TTT ATC GAT ACT ATC AAT AAA CCT GTA AAT GCA GCA CTA TTT ATA GAT 1776 Phe Ile Asp Thr Ile Asn Lys Pro Val Asn Ala Ala Leu Phe Ile Asp 580 585 590 TGG ATA AGC AAA GTA ATA AGA GAT TTT ACC ACT GAA GCT ACA CAA AAA 1824 Trp Ile Ser Lys Val Ile Arg Asp Phe Thr Thr Glu Ala Thr Gln Lys 595 600 605 AGT ACT GTT GAT AAG ATT GCA GAC ATA TCT TTA ATT GTA CCC TAT GTA 1872 Ser Thr Val Asp Lys Ile Ala Asp Ile Ser Leu Ile Val Pro Tyr Val 610 615 620 GGT CTT GCT TTG AAT ATA ATT ATT GAG GCA GAA AAA GGA AAT TTT GAG 1920 Gly Leu Ala Leu Asn Ile Ile Ile Glu Ala Glu Lys Gly Asn Phe Glu 625 630 635 640 GAG GCA TTT GAA TTA TTA GGA GTG GGT ATT TTA TTA GAA TTT GTG CCA 1968 Glu Ala Phe Glu Leu Leu Gly Val Gly Ile Leu Leu Glu Phe Val Pro 645 650 655 GAA CTT ACA ATT CCT GTA ATT TTA GTG TTT ACG ATA AAA TCC TAT ATA 2016 Glu Leu Thr Ile Pro Val Ile Leu Val Phe Thr Ile Lys Ser Tyr Ile 660 665 670 GAT TCA TAT GAG AAT AAA AAT AAA GCA ATT AAA GCA ATA AAT AAT TCA 2064 Asp Ser Tyr Glu Asn Lys Asn Lys Ala Ile Lys Ala Ile Asn Asn Ser 675 680 685 TTA ATC GAA AGA GAA GCA AAG TGG AAA GAA ATA TAT AGT TGG ATA GTA 2112 Leu Ile Glu Arg Glu Ala Lys Trp Lys Glu Ile Tyr Ser Trp Ile Val 690 695 700 TCA AAT TGG CTT ACT AGA ATT AAT ACT CAA TTT AAT AAA AGA AAA GAG 2160 Ser Asn Trp Leu Thr Arg Ile Asn Thr Gln Phe Asn Lys Arg Lys Glu 705 710 715 720 CAA ATG TAT CAG GCT TTA CAA AAT CAA GTA GAT GCA ATA AAA ACA GCA 2208 Gln Met Tyr Gln Ala Leu Gln Asn Gln Val Asp Ala Ile Lys Thr Ala 725 730 735 ATA GAA TAT AAA TAT AAT AAT TAT ACT TCA GAT GAG AAA AAT AGA CTT 2256 Ile Glu Tyr Lys Tyr Asn Asn Tyr Thr Ser Asp Glu Lys Asn Arg Leu 740 745 750 GAA TCT GAA TAT AAT ATC AAT AAT ATA GAA GAA GAA TTG AAT AAA AAA 2304 Glu Ser Glu Tyr Asn Ile Asn Asn Ile Glu Glu Glu Leu Asn Lys Lys 755 760 765 GTT TCT TTA GCA ATG AAA AAT ATA GAA AGA TTT ATG ACA GAA AGT TCT 2352 Val Ser Leu Ala Met Lys Asn Ile Glu Arg Phe Met Thr Glu Ser Ser 770 775 780 ATA TCT TAT TTA ATG AAA TTA ATA AAT GAA GCC AAA GTT GGT AAA TTA 2400 Ile Ser Tyr Leu Met Lys Leu Ile Asn Glu Ala Lys Val Gly Lys Leu 785 790 795 800 AAA AAA TAT GAT AAC CAT GTT AAG AGC GAT TTA TTA AAC TAT ATT CTC 2448 Lys Lys Tyr Asp Asn His Val Lys Ser Asp Leu Leu Asn Tyr Ile Leu 805 810 815 GAC CAT AGA TCA ATC TTA GGA GAG CAG ACA AAT GAA TTA AGT GAT TTG 2496 Asp His Arg Ser Ile Leu Gly Glu Gln Thr Asn Glu Leu Ser Asp Leu 820 825 830 GTG ACT AGT ACT TTG AAT AGT AGT ATT CCA TTT GAA CTT TCT TCA TAT 2544 Val Thr Ser Thr Leu Asn Ser Ser Ile Pro Phe Glu Leu Ser Ser Tyr 835 840 845 ACT AAT GAT AAA ATT CTA ATT ATA TAT TTT AAT AGA TTA TAT AAA AAA 2592 Thr Asn Asp Lys Ile Leu Ile Ile Tyr Phe Asn Arg Leu Tyr Lys Lys 850 855 860 ATT AAA GAT AGT TCT ATT TTA GAT ATG CGA TAT GAA AAT AAT AAA TTT 2640 Ile Lys Asp Ser Ser Ile Leu Asp Met Arg Tyr Glu Asn Asn Lys Phe 865 870 875 880 ATA GAT ATC TCT GGA TAT GGT TCA AAT ATA AGC ATT AAT GGA AAC GTA 2688 Ile Asp Ile Ser Gly Tyr Gly Ser Asn Ile Ser Ile Asn Gly Asn Val 885 890 895 TAT ATT TAT TCA ACA AAT AGA AAT CAA TTT GGA ATA TAT AAT AGT AGG 2736 Tyr Ile Tyr Ser Thr Asn Arg Asn Gln Phe Gly Ile Tyr Asn Ser Arg 900 905 910 CTT AGT GAA GTT AAT ATA GCT CAA AAT AAT GAT ATT ATA TAC AAT AGT 2784 Leu Ser Glu Val Asn Ile Ala Gln Asn Asn Asp Ile Ile Tyr Asn Ser 915 920 925 AGA TAT CAA AAT TTT AGT ATT AGT TTC TGG GTA AGG ATT CCT AAA CAC 2832 Arg Tyr Gln Asn Phe Ser Ile Ser Phe Trp Val Arg Ile Pro Lys His 930 935 940 TAC AAA CCT ATG AAT CAT AAT CGG GAA TAC ACT ATA ATA AAT TGT ATG 2880 Tyr Lys Pro Met Asn His Asn Arg Glu Tyr Thr Ile Ile Asn Cys Met 945 950 955 960 GGG AAT AAT AAT TCG GGA TGG AAA ATA TCA CTT AGA ACT GTT AGA GAT 2928 Gly Asn Asn Asn Ser Gly Trp Lys Ile Ser Leu Arg Thr Val Arg Asp 965 970 975 TGT GAA ATA ATT TGG ACT TTA CAA GAT ACT TCT GGA AAT AAG GAA AAT 2976 Cys Glu Ile Ile Trp Thr Leu Gln Asp Thr Ser Gly Asn Lys Glu Asn 980 985 990 TTA ATT TTT AGG TAT GAA GAA CTT AAT AGG ATA TCT AAT TAT ATA AAT 3024 Leu Ile Phe Arg Tyr Glu Glu Leu Asn Arg Ile Ser Asn Tyr Ile Asn 995 1000 1005 AAA TGG ATT TTT GTA ACT ATT ACT AAT AAT AGA TTA GGC AAT TCT AGA 3072 Lys Trp Ile Phe Val Thr Ile Thr Asn Asn Arg Leu Gly Asn Ser Arg 1010 1015 1020 ATT TAC ATC AAT GGA AAT TTA ATA GTT GAA AAA TCA ATT TCG AAT TTA 3120 Ile Tyr Ile Asn Gly Asn Leu Ile Val Glu Lys Ser Ile Ser Asn Leu 1025 1030 1035 1040 GGT GAT ATT CAT GTT AGT GAT AAT ATA TTA TTT AAA ATT GTT GGT TGT 3168 Gly Asp Ile His Val Ser Asp Asn Ile Leu Phe Lys Ile Val Gly Cys 1045 1050 1055 GAT GAT GAA ACG TAT GTT GGT ATA AGA TAT TTT AAA GTT TTT AAT ACG 3216 Asp Asp Glu Thr Tyr Val Gly Ile Arg Tyr Phe Lys Val Phe Asn Thr 1060 1065 1070 GAA TTA GAT AAA ACA GAA ATT GAG ACT TTA TAT AGT AAT GAG CCA GAT 3264 Glu Leu Asp Lys Thr Glu Ile Glu Thr Leu Tyr Ser Asn Glu Pro Asp 1075 1080 1085 CCA AGT ATC TTA AAA AAC TAT TGG GGA AAT TAT TTG CTA TAT AAT AAA 3312 Pro Ser Ile Leu Lys Asn Tyr Trp Gly Asn Tyr Leu Leu Tyr Asn Lys 1090 1095 1100 AAA TAT TAT TTA TTC AAT TTA CTA AGA AAA GAT AAG TAT ATT ACT CTG 3360 Lys Tyr Tyr Leu Phe Asn Leu Leu Arg Lys Asp Lys Tyr Ile Thr Leu 1105 1110 1115 1120 AAT TCA GGC ATT TTA AAT ATT AAT CAA CAA AGA GGT GTT ACT GAA GGC 3408 Asn Ser Gly Ile Leu Asn Ile Asn Gln Gln Arg Gly Val Thr Glu Gly 1125 1130 1135 TCT GTT TTT TTG AAC TAT AAA TTA TAT GAA GGA GTA GAA GTC ATT ATA 3456 Ser Val Phe Leu Asn Tyr Lys Leu Tyr Glu Gly Val Glu Val Ile Ile 1140 1145 1150 AGA AAA AAT GGT CCT ATA GAT ATA TCT AAT ACA GAT AAT TTT GTT AGA 3504 Arg Lys Asn Gly Pro Ile Asp Ile Ser Asn Thr Asp Asn Phe Val Arg 1155 1160 1165 AAA AAC GAT CTA GCA TAC ATT AAT GTA GTA GAT CGT GGT GTA GAA TAT 3552 Lys Asn Asp Leu Ala Tyr Ile Asn Val Val Asp Arg Gly Val Glu Tyr 1170 1175 1180 CGG TTA TAT GCT GAT ACA AAA TCA GAG AAA GAG AAA ATA ATA AGA ACA 3600 Arg Leu Tyr Ala Asp Thr Lys Ser Glu Lys Glu Lys Ile Ile Arg Thr 1185 1190 1195 1200 TCT AAT CTA AAC GAT AGC TTA GGT CAA ATT ATA GTT ATG GAT TCA ATA 3648 Ser Asn Leu Asn Asp Ser Leu Gly Gln Ile Ile Val Met Asp Ser Ile 1205 1210 1215 GGA AAT AAT TGC ACA ATG AAT TTT CAA AAC AAT AAT GGG AGC AAT ATA 3696 Gly Asn Asn Cys Thr Met Asn Phe Gln Asn Asn Asn Gly Ser Asn Ile 1220 1225 1230 GGA TTA CTA GGT TTT CAT TCA AAT AAT TTG GTT GCT AGT AGT TGG TAT 3744 Gly Leu Leu Gly Phe His Ser Asn Asn Leu Val Ala Ser Ser Trp Tyr 1235 1240 1245 TAT AAC AAT ATA CGA AGA AAT ACT AGC AGT AAT GGA TGC TTT TGG AGT 3792 Tyr Asn Asn Ile Arg Arg Asn Thr Ser Ser Asn Gly Cys Phe Trp Ser 1250 1255 1260 TCT ATT TCT AAA GAG AAT GGA TGG AAA GAA TGA 3825 Ser Ile Ser Lys Glu Asn Gly Trp Lys Glu 1265 1270

71 Met Pro Val Ala Ile Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asp 1 5 10 15 Asp Thr Ile Leu Tyr Met Gln Ile Pro Tyr Glu Glu Lys Ser Lys Lys 20 25 30 Tyr Tyr Lys Ala Phe Glu Ile Met Arg Asn Val Trp Ile Ile Pro Glu 35 40 45 Arg Asn Thr Ile Gly Thr Asn Pro Ser Asp Phe Asp Pro Pro Ala Ser 50 55 60 Leu Lys Asn Gly Ser Ser Ala Tyr Tyr Asp Pro Asn Tyr Leu Thr Thr 65 70 75 80 Asp Ala Glu Lys Asp Arg Tyr Leu Lys Thr Thr Ile Lys Leu Phe Lys 85 90 95 Arg Ile Asn Ser Asn Pro Ala Gly Lys Val Leu Leu Gln Glu Ile Ser 100 105 110 Tyr Ala Lys Pro Tyr Leu Gly Asn Asp His Thr Pro Ile Asp Glu Phe 115 120 125 Ser Pro Val Thr Arg Thr Thr Ser Val Asn Ile Lys Leu Ser Thr Asn 130 135 140 Val Glu Ser Ser Met Leu Leu Asn Leu Leu Val Leu Gly Ala Gly Pro 145 150 155 160 Asp Ile Phe Glu Ser Cys Cys Tyr Pro Val Arg Lys Leu Ile Asp Pro 165 170 175 Asp Val Val Tyr Asp Pro Ser Asn Tyr Gly Phe Gly Ser Ile Asn Ile 180 185 190 Val Thr Phe Ser Pro Glu Tyr Glu Tyr Thr Phe Asn Asp Ile Ser Gly 195 200 205 Gly His Asn Ser Ser Thr Glu Ser Phe Ile Ala Asp Pro Ala Ile Ser 210 215 220 Leu Ala His Glu Leu Ile His Ala Leu His Gly Leu Tyr Gly Ala Arg 225 230 235 240 Gly Val Thr Tyr Glu Glu Thr Ile Glu Val Lys Gln Ala Pro Leu Met 245 250 255 Ile Ala Glu Lys Pro Ile Arg Leu Glu Glu Phe Leu Thr Phe Gly Gly 260 265 270 Gln Asp Leu Asn Ile Ile Thr Ser Ala Met Lys Glu Lys Ile Tyr Asn 275 280 285 Asn Leu Leu Ala Asn Tyr Glu Lys Ile Ala Thr Arg Leu Ser Glu Val 290 295 300 Asn Ser Ala Pro Pro Glu Tyr Asp Ile Asn Glu Tyr Lys Asp Tyr Phe 305 310 315 320 Gln Trp Lys Tyr Gly Leu Asp Lys Asn Ala Asp Gly Ser Tyr Thr Val 325 330 335 Asn Glu Asn Lys Phe Asn Glu Ile Tyr Lys Lys Leu Tyr Ser Phe Thr 340 345 350 Glu Ser Asp Leu Ala Asn Lys Phe Lys Val Lys Cys Arg Asn Thr Tyr 355 360 365 Phe Ile Lys Tyr Glu Phe Leu Lys Val Pro Asn Leu Leu Asp Asp Asp 370 375 380 Ile Tyr Thr Val Ser Glu Gly Phe Asn Ile Gly Asn Leu Ala Val Asn 385 390 395 400 Asn Arg Gly Gln Ser Ile Lys Leu Asn Pro Lys Ile Ile Asp Ser Ile 405 410 415 Pro Asp Lys Gly Leu Val Glu Lys Ile Val Lys Phe Cys Lys Ser Val 420 425 430 Ile Pro Arg Lys Gly Thr Lys Ala Pro Pro Arg Leu Cys Ile Arg Val 435 440 445 Asn Asn Ser Glu Leu Phe Phe Val Ala Ser Glu Ser Ser Tyr Asn Glu 450 455 460 Asn Asp Ile Asn Thr Pro Lys Glu Ile Asp Asp Thr Thr Asn Leu Asn 465 470 475 480 Asn Asn Tyr Arg Asn Asn Leu Asp Glu Val Ile Leu Asp Tyr Asn Ser 485 490 495 Gln Thr Ile Pro Gln Ile Ser Asn Arg Thr Leu Asn Thr Leu Val Gln 500 505 510 Asp Asn Ser Tyr Val Pro Arg Tyr Asp Ser Asn Gly Thr Ser Glu Ile 515 520 525 Glu Glu Tyr Asp Val Val Asp Phe Asn Val Phe Phe Tyr Leu His Ala 530 535 540 Gln Lys Val Pro Glu Gly Glu Thr Asn Ile Ser Leu Thr Ser Ser Ile 545 550 555 560 Asp Thr Ala Leu Leu Glu Glu Ser Lys Asp Ile Phe Phe Ser Ser Glu 565 570 575 Phe Ile Asp Thr Ile Asn Lys Pro Val Asn Ala Ala Leu Phe Ile Asp 580 585 590 Trp Ile Ser Lys Val Ile Arg Asp Phe Thr Thr Glu Ala Thr Gln Lys 595 600 605 Ser Thr Val Asp Lys Ile Ala Asp Ile Ser Leu Ile Val Pro Tyr Val 610 615 620 Gly Leu Ala Leu Asn Ile Ile Ile Glu Ala Glu Lys Gly Asn Phe Glu 625 630 635 640 Glu Ala Phe Glu Leu Leu Gly Val Gly Ile Leu Leu Glu Phe Val Pro 645 650 655 Glu Leu Thr Ile Pro Val Ile Leu Val Phe Thr Ile Lys Ser Tyr Ile 660 665 670 Asp Ser Tyr Glu Asn Lys Asn Lys Ala Ile Lys Ala Ile Asn Asn Ser 675 680 685 Leu Ile Glu Arg Glu Ala Lys Trp Lys Glu Ile Tyr Ser Trp Ile Val 690 695 700 Ser Asn Trp Leu Thr Arg Ile Asn Thr Gln Phe Asn Lys Arg Lys Glu 705 710 715 720 Gln Met Tyr Gln Ala Leu Gln Asn Gln Val Asp Ala Ile Lys Thr Ala 725 730 735 Ile Glu Tyr Lys Tyr Asn Asn Tyr Thr Ser Asp Glu Lys Asn Arg Leu 740 745 750 Glu Ser Glu Tyr Asn Ile Asn Asn Ile Glu Glu Glu Leu Asn Lys Lys 755 760 765 Val Ser Leu Ala Met Lys Asn Ile Glu Arg Phe Met Thr Glu Ser Ser 770 775 780 Ile Ser Tyr Leu Met Lys Leu Ile Asn Glu Ala Lys Val Gly Lys Leu 785 790 795 800 Lys Lys Tyr Asp Asn His Val Lys Ser Asp Leu Leu Asn Tyr Ile Leu 805 810 815 Asp His Arg Ser Ile Leu Gly Glu Gln Thr Asn Glu Leu Ser Asp Leu 820 825 830 Val Thr Ser Thr Leu Asn Ser Ser Ile Pro Phe Glu Leu Ser Ser Tyr 835 840 845 Thr Asn Asp Lys Ile Leu Ile Ile Tyr Phe Asn Arg Leu Tyr Lys Lys 850 855 860 Ile Lys Asp Ser Ser Ile Leu Asp Met Arg Tyr Glu Asn Asn Lys Phe 865 870 875 880 Ile Asp Ile Ser Gly Tyr Gly Ser Asn Ile Ser Ile Asn Gly Asn Val 885 890 895 Tyr Ile Tyr Ser Thr Asn Arg Asn Gln Phe Gly Ile Tyr Asn Ser Arg 900 905 910 Leu Ser Glu Val Asn Ile Ala Gln Asn Asn Asp Ile Ile Tyr Asn Ser 915 920 925 Arg Tyr Gln Asn Phe Ser Ile Ser Phe Trp Val Arg Ile Pro Lys His 930 935 940 Tyr Lys Pro Met Asn His Asn Arg Glu Tyr Thr Ile Ile Asn Cys Met 945 950 955 960 Gly Asn Asn Asn Ser Gly Trp Lys Ile Ser Leu Arg Thr Val Arg Asp 965 970 975 Cys Glu Ile Ile Trp Thr Leu Gln Asp Thr Ser Gly Asn Lys Glu Asn 980 985 990 Leu Ile Phe Arg Tyr Glu Glu Leu Asn Arg Ile Ser Asn Tyr Ile Asn 995 1000 1005 Lys Trp Ile Phe Val Thr Ile Thr Asn Asn Arg Leu Gly Asn Ser Arg 1010 1015 1020 Ile Tyr Ile Asn Gly Asn Leu Ile Val Glu Lys Ser Ile Ser Asn Leu 1025 1030 1035 1040 Gly Asp Ile His Val Ser Asp Asn Ile Leu Phe Lys Ile Val Gly Cys 1045 1050 1055 Asp Asp Glu Thr Tyr Val Gly Ile Arg Tyr Phe Lys Val Phe Asn Thr 1060 1065 1070 Glu Leu Asp Lys Thr Glu Ile Glu Thr Leu Tyr Ser Asn Glu Pro Asp 1075 1080 1085 Pro Ser Ile Leu Lys Asn Tyr Trp Gly Asn Tyr Leu Leu Tyr Asn Lys 1090 1095 1100 Lys Tyr Tyr Leu Phe Asn Leu Leu Arg Lys Asp Lys Tyr Ile Thr Leu 1105 1110 1115 1120 Asn Ser Gly Ile Leu Asn Ile Asn Gln Gln Arg Gly Val Thr Glu Gly 1125 1130 1135 Ser Val Phe Leu Asn Tyr Lys Leu Tyr Glu Gly Val Glu Val Ile Ile 1140 1145 1150 Arg Lys Asn Gly Pro Ile Asp Ile Ser Asn Thr Asp Asn Phe Val Arg 1155 1160 1165 Lys Asn Asp Leu Ala Tyr Ile Asn Val Val Asp Arg Gly Val Glu Tyr 1170 1175 1180 Arg Leu Tyr Ala Asp Thr Lys Ser Glu Lys Glu Lys Ile Ile Arg Thr 1185 1190 1195 1200 Ser Asn Leu Asn Asp Ser Leu Gly Gln Ile Ile Val Met Asp Ser Ile 1205 1210 1215 Gly Asn Asn Cys Thr Met Asn Phe Gln Asn Asn Asn Gly Ser Asn Ile 1220 1225 1230 Gly Leu Leu Gly Phe His Ser Asn Asn Leu Val Ala Ser Ser Trp Tyr 1235 1240 1245 Tyr Asn Asn Ile Arg Arg Asn Thr Ser Ser Asn Gly Cys Phe Trp Ser 1250 1255 1260 Ser Ile Ser Lys Glu Asn Gly Trp Lys Glu 1265 1270

72 AGATCTCGAT CCCGCGAAAT TAATACGACT CACTATAGGG GAATTGTGAG CGGATAACAA 60 TTCCCCTCTA GAAATAATTT TGTTTAACTT TAAGAAGGAG ATATACC ATG GGC CAT 116 Met Gly His 1 CAT CAT CAT CAT CAT CAT CAT CAT CAC AGC AGC GGC CAT ATC GAA GGT 164 His His His His His His His His His Ser Ser Gly His Ile GluGly 5 10 15 CGT CAT ATG GCT AGC ATG GCT ATT CTA ATT ATA TAT TTT AAT AGA TTA 212 Arg His Met Ala Ser Met Ala Ile Leu Ile Ile Tyr Phe Asn Arg Leu 20 25 30 35 TAT AAA AAA ATT AAA GAT AGT TCT ATT TTA GAT ATG CGA TAT GAA AAT 260 Tyr Lys Lys Ile Lys Asp Ser Ser Ile Leu Asp Met Arg Tyr Glu Asn 40 45 50 AAT AAA TTT ATA GAT ATC TCT GGA TAT GGT TCA AAT ATA AGC ATT AAT 308 Asn Lys Phe Ile Asp Ile Ser Gly Tyr Gly Ser Asn Ile Ser Ile Asn 55 60 65 GGA AAC GTA TAT ATT TAT TCA ACA AAT AGA AAT CAA TTT GGA ATA TAT 356 Gly Asn Val Tyr Ile Tyr Ser Thr Asn Arg Asn Gln Phe Gly Ile Tyr 70 75 80 AAT AGT AGG CTT AGT GAA GTT AAT ATA GCT CAA AAT AAT GAT ATT ATA 404 Asn Ser Arg Leu Ser Glu Val Asn Ile Ala Gln Asn Asn Asp Ile Ile 85 90 95 TAC AAT AGT AGA TAT CAA AAT TTT AGT ATT AGT TTC TGG GTA AGG ATT 452 Tyr Asn Ser Arg Tyr Gln Asn Phe Ser Ile Ser Phe Trp Val Arg Ile 100 105 110 115 CCT AAA CAC TAC AAA CCT ATG AAT CAT AAT CGG GAA TAC ACT ATA ATA 500 Pro Lys His Tyr Lys Pro Met Asn His Asn Arg Glu Tyr Thr Ile Ile 120 125 130 AAT TGT ATG GGG AAT AAT AAT TCG GGA TGG AAA ATA TCA CTT AGA ACT 548 Asn Cys Met Gly Asn Asn Asn Ser Gly Trp Lys Ile Ser Leu Arg Thr 135 140 145 GTT AGA GAT TGT GAA ATA ATT TGG ACT TTA CAA GAT ACT TCT GGA AAT 596 Val Arg Asp Cys Glu Ile Ile Trp Thr Leu Gln Asp Thr Ser Gly Asn 150 155 160 AAG GAA AAT TTA ATT TTT AGG TAT GAA GAA CTT AAT AGG ATA TCT AAT 644 Lys Glu Asn Leu Ile Phe Arg Tyr Glu Glu Leu Asn Arg Ile Ser Asn 165 170 175 TAT ATA AAT AAA TGG ATT TTT GTA ACT ATT ACT AAT AAT AGA TTA GGC 692 Tyr Ile Asn Lys Trp Ile Phe Val Thr Ile Thr Asn Asn Arg Leu Gly 180 185 190 195 AAT TCT AGA ATT TAC ATC AAT GGA AAT TTA ATA GTT GAA AAA TCA ATT 740 Asn Ser Arg Ile Tyr Ile Asn Gly Asn Leu Ile Val Glu Lys Ser Ile 200 205 210 TCG AAT TTA GGT GAT ATT CAT GTT AGT GAT AAT ATA TTA TTT AAA ATT 788 Ser Asn Leu Gly Asp Ile His Val Ser Asp Asn Ile Leu Phe Lys Ile 215 220 225 GTT GGT TGT GAT GAT GAA ACG TAT GTT GGT ATA AGA TAT TTT AAA GTT 836 Val Gly Cys Asp Asp Glu Thr Tyr Val Gly Ile Arg Tyr Phe Lys Val 230 235 240 TTT AAT ACG GAA TTA GAT AAA ACA GAA ATT GAG ACT TTA TAT AGT AAT 884 Phe Asn Thr Glu Leu Asp Lys Thr Glu Ile Glu Thr Leu Tyr Ser Asn 245 250 255 GAG CCA GAT CCA AGT ATC TTA AAA AAC TAT TGG GGA AAT TAT TTG CTA 932 Glu Pro Asp Pro Ser Ile Leu Lys Asn Tyr Trp Gly Asn Tyr Leu Leu 260 265 270 275 TAT AAT AAA AAA TAT TAT TTA TTC AAT TTA CTA AGA AAA GAT AAG TAT 980 Tyr Asn Lys Lys Tyr Tyr Leu Phe Asn Leu Leu Arg Lys Asp Lys Tyr 280 285 290 ATT ACT CTG AAT TCA GGC ATT TTA AAT ATT AAT CAA CAA AGA GGT GTT 1028 Ile Thr Leu Asn Ser Gly Ile Leu Asn Ile Asn Gln Gln Arg Gly Val 295 300 305 ACT GAA GGC TCT GTT TTT TTG AAC TAT AAA TTA TAT GAA GGA GTA GAA 1076 Thr Glu Gly Ser Val Phe Leu Asn Tyr Lys Leu Tyr Glu Gly Val Glu 310 315 320 GTC ATT ATA AGA AAA AAT GGT CCT ATA GAT ATA TCT AAT ACA GAT AAT 1124 Val Ile Ile Arg Lys Asn Gly Pro Ile Asp Ile Ser Asn Thr Asp Asn 325 330 335 TTT GTT AGA AAA AAC GAT CTA GCA TAC ATT AAT GTA GTA GAT CGT GGT 1172 Phe Val Arg Lys Asn Asp Leu Ala Tyr Ile Asn Val Val Asp Arg Gly 340 345 350 355 GTA GAA TAT CGG TTA TAT GCT GAT ACA AAA TCA GAG AAA GAG AAA ATA 1220 Val Glu Tyr Arg Leu Tyr Ala Asp Thr Lys Ser Glu Lys Glu Lys Ile 360 365 370 ATA AGA ACA TCT AAT CTA AAC GAT AGC TTA GGT CAA ATT ATA GTT ATG 1268 Ile Arg Thr Ser Asn Leu Asn Asp Ser Leu Gly Gln Ile Ile Val Met 375 380 385 GAT TCA ATA GGA AAT AAT TGC ACA ATG AAT TTT CAA AAC AAT AAT GGG 1316 Asp Ser Ile Gly Asn Asn Cys Thr Met Asn Phe Gln Asn Asn Asn Gly 390 395 400 AGC AAT ATA GGA TTA CTA GGT TTT CAT TCA AAT AAT TTG GTT GCT AGT 1364 Ser Asn Ile Gly Leu Leu Gly Phe His Ser Asn Asn Leu Val Ala Ser 405 410 415 AGT TGG TAT TAT AAC AAT ATA CGA AGA AAT ACT AGC AGT AAT GGA TGC 1412 Ser Trp Tyr Tyr Asn Asn Ile Arg Arg Asn Thr Ser Ser Asn Gly Cys 420 425 430 435 TTT TGG AGT TCT ATT TCT AAA GAG AAT GGA TGG AAA GAA TGAAAGCTT 1460 Phe Trp Ser Ser Ile Ser Lys Glu Asn Gly Trp Lys Glu 440 445

73 Met Gly His His His His His His His His His His Ser Ser Gly His 1 5 10 15 Ile Glu Gly Arg His Met Ala Ser Met Ala Ile Leu Ile Ile TyrPhe 20 25 30 Asn Arg Leu Tyr Lys Lys Ile Lys Asp Ser Ser Ile Leu Asp Met Arg 35 40 45 Tyr Glu Asn Asn Lys Phe Ile Asp Ile Ser Gly Tyr Gly Ser Asn Ile 50 55 60 Ser Ile Asn Gly Asn Val Tyr Ile Tyr Ser Thr Asn Arg Asn Gln Phe 65 70 75 80 Gly Ile Tyr Asn Ser Arg Leu Ser Glu Val Asn Ile Ala Gln Asn Asn 85 90 95 Asp Ile Ile Tyr Asn Ser Arg Tyr Gln Asn Phe Ser Ile Ser Phe Trp 100 105 110 Val Arg Ile Pro Lys His Tyr Lys Pro Met Asn His Asn Arg Glu Tyr 115 120 125 Thr Ile Ile Asn Cys Met Gly Asn Asn Asn Ser Gly Trp Lys Ile Ser 130 135 140 Leu Arg Thr Val Arg Asp Cys Glu Ile Ile Trp Thr Leu Gln Asp Thr 145 150 155 160 Ser Gly Asn Lys Glu Asn Leu Ile Phe Arg Tyr Glu Glu Leu Asn Arg 165 170 175 Ile Ser Asn Tyr Ile Asn Lys Trp Ile Phe Val Thr Ile Thr Asn Asn 180 185 190 Arg Leu Gly Asn Ser Arg Ile Tyr Ile Asn Gly Asn Leu Ile Val Glu 195 200 205 Lys Ser Ile Ser Asn Leu Gly Asp Ile His Val Ser Asp Asn Ile Leu 210 215 220 Phe Lys Ile Val Gly Cys Asp Asp Glu Thr Tyr Val Gly Ile Arg Tyr 225 230 235 240 Phe Lys Val Phe Asn Thr Glu Leu Asp Lys Thr Glu Ile Glu Thr Leu 245 250 255 Tyr Ser Asn Glu Pro Asp Pro Ser Ile Leu Lys Asn Tyr Trp Gly Asn 260 265 270 Tyr Leu Leu Tyr Asn Lys Lys Tyr Tyr Leu Phe Asn Leu Leu Arg Lys 275 280 285 Asp Lys Tyr Ile Thr Leu Asn Ser Gly Ile Leu Asn Ile Asn Gln Gln 290 295 300 Arg Gly Val Thr Glu Gly Ser Val Phe Leu Asn Tyr Lys Leu Tyr Glu 305 310 315 320 Gly Val Glu Val Ile Ile Arg Lys Asn Gly Pro Ile Asp Ile Ser Asn 325 330 335 Thr Asp Asn Phe Val Arg Lys Asn Asp Leu Ala Tyr Ile Asn Val Val 340 345 350 Asp Arg Gly Val Glu Tyr Arg Leu Tyr Ala Asp Thr Lys Ser Glu Lys 355 360 365 Glu Lys Ile Ile Arg Thr Ser Asn Leu Asn Asp Ser Leu Gly Gln Ile 370 375 380 Ile Val Met Asp Ser Ile Gly Asn Asn Cys Thr Met Asn Phe Gln Asn 385 390 395 400 Asn Asn Gly Ser Asn Ile Gly Leu Leu Gly Phe His Ser Asn Asn Leu 405 410 415 Val Ala Ser Ser Trp Tyr Tyr Asn Asn Ile Arg Arg Asn Thr Ser Ser 420 425 430 Asn Gly Cys Phe Trp Ser Ser Ile Ser Lys Glu Asn Gly Trp Lys Glu 435 440 445

74 CGCCATGGCT ATTCTAATTA TATATTTTAA TAG 33

75 GCAAGCTTTC ATTCTTTCCA TCCATTCTC 29

76 ATG CCA GTT AAT ATA AAA AAC TTT AAT TAT AAT GAC CCT ATT AAT AAT 48 Met Pro Val Asn Ile Lys Asn Phe Asn Tyr Asn Asp Pro Ile Asn Asn 1 5 10 15 GAT GAC ATT ATT ATG ATG GAA CCA TTC AAT GAC CCA GGG CCA GGA ACA 96 Asp Asp Ile Ile Met Met Glu Pro Phe Asn Asp Pro Gly Pro Gly Thr 20 25 30 TAT TAT AAA GCT TTT AGG ATT ATA GAT CGT ATT TGG ATA GTA CCA GAA 144 Tyr Tyr Lys Ala Phe Arg Ile Ile Asp Arg Ile Trp Ile Val Pro Glu 35 40 45 AGG TTT ACT TAT GGA TTT CAA CCT GAC CAA TTT AAT GCC AGT ACA GGA 192 Arg Phe Thr Tyr Gly Phe Gln Pro Asp Gln Phe Asn Ala Ser Thr Gly 50 55 60 GTT TTT AGT AAA GAT GTC TAC GAA TAT TAC GAT CCA ACT TAT TTA AAA 240 Val Phe Ser Lys Asp Val Tyr Glu Tyr Tyr Asp Pro Thr Tyr Leu Lys 65 70 75 80 ACC GAT GCT GAA AAA GAT AAA TTT TTA AAA ACA ATG ATT AAA TTA TTT 288 Thr Asp Ala Glu Lys Asp Lys Phe Leu Lys Thr Met Ile Lys Leu Phe 85 90 95 AAT AGA ATT AAT TCA AAA CCA TCA GGA CAG AGA TTA CTG GAT ATG ATA 336 Asn Arg Ile Asn Ser Lys Pro Ser Gly Gln Arg Leu Leu Asp Met Ile 100 105 110 GTA GAT GCT ATA CCT TAT CTT GGA AAT GCA TCT ACA CCG CCC GAC AAA 384 Val Asp Ala Ile Pro Tyr Leu Gly Asn Ala Ser Thr Pro Pro Asp Lys 115 120 125 TTT GCA GCA AAT GTT GCA AAT GTA TCT ATT AAT AAA AAA ATT ATC CAA 432 Phe Ala Ala Asn Val Ala Asn Val Ser Ile Asn Lys Lys Ile Ile Gln 130 135 140 CCT GGA GCT GAA GAT CAA ATA AAA GGT TTA ATG ACA AAT TTA ATA ATA 480 Pro Gly Ala Glu Asp Gln Ile Lys Gly Leu Met Thr Asn Leu Ile Ile 145 150 155 160 TTT GGA CCA GGA CCA GTT CTA AGT GAT AAT TTT ACT GAT AGT ATG ATT 528 Phe Gly Pro Gly Pro Val Leu Ser Asp Asn Phe Thr Asp Ser Met Ile 165 170 175 ATG AAT GGC CAT TCC CCA ATA TCA GAA GGA TTT GGT GCA AGA ATG ATG 576 Met Asn Gly His Ser Pro Ile Ser Glu Gly Phe Gly Ala Arg Met Met 180 185 190 ATA AGA TTT TGT CCT AGT TGT TTA AAT GTA TTT AAT AAT GTT CAG GAA 624 Ile Arg Phe Cys Pro Ser Cys Leu Asn Val Phe Asn Asn Val Gln Glu 195 200 205 AAT AAA GAT ACA TCT ATA TTT AGT AGA CGC GCG TAT TTT GCA GAT CCA 672 Asn Lys Asp Thr Ser Ile Phe Ser Arg Arg Ala Tyr Phe Ala Asp Pro 210 215 220 GCT CTA ACG TTA ATG CAT GAA CTT ATA CAT GTG TTA CAT GGA TTA TAT 720 Ala Leu Thr Leu Met His Glu Leu Ile His Val Leu His Gly Leu Tyr 225 230 235 240 GGA ATT AAG ATA AGT AAT TTA CCA ATT ACT CCA AAT ACA AAA GAA TTT 768 Gly Ile Lys Ile Ser Asn Leu Pro Ile Thr Pro Asn Thr Lys Glu Phe 245 250 255 TTC ATG CAA CAT AGC GAT CCT GTA CAA GCA GAA GAA CTA TAT ACA TTC 816 Phe Met Gln His Ser Asp Pro Val Gln Ala Glu Glu Leu Tyr Thr Phe 260 265 270 GGA GGA CAT GAT CCT AGT GTT ATA AGT CCT TCT ACG GAT ATG AAT ATT 864 Gly Gly His Asp Pro Ser Val Ile Ser Pro Ser Thr Asp Met Asn Ile 275 280 285 TAT AAT AAA GCG TTA CAA AAT TTT CAA GAT ATA GCT AAT AGG CTT AAT 912 Tyr Asn Lys Ala Leu Gln Asn Phe Gln Asp Ile Ala Asn Arg Leu Asn 290 295 300 ATT GTT TCA AGT GCC CAA GGG AGT GGA ATT GAT ATT TCC TTA TAT AAA 960 Ile Val Ser Ser Ala Gln Gly Ser Gly Ile Asp Ile Ser Leu Tyr Lys 305 310 315 320 CAA ATA TAT AAA AAT AAA TAT GAT TTT GTT GAA GAT CCT AAT GGA AAA 1008 Gln Ile Tyr Lys Asn Lys Tyr Asp Phe Val Glu Asp Pro Asn Gly Lys 325 330 335 TAT AGT GTA GAT AAG GAT AAG TTT GAT AAA TTA TAT AAG GCC TTA ATG 1056 Tyr Ser Val Asp Lys Asp Lys Phe Asp Lys Leu Tyr Lys Ala Leu Met 340 345 350 TTT GGC TTT ACT GAA ACT AAT CTA GCT GGT GAA TAT GGA ATA AAA ACT 1104 Phe Gly Phe Thr Glu Thr Asn Leu Ala Gly Glu Tyr Gly Ile Lys Thr 355 360 365 AGG TAT TCT TAT TTT AGT GAA TAT TTG CCA CCG ATA AAA ACT GAA AAA 1152 Arg Tyr Ser Tyr Phe Ser Glu Tyr Leu Pro Pro Ile Lys Thr Glu Lys 370 375 380 TTG TTA GAC AAT ACA ATT TAT ACT CAA AAT GAA GGC TTT AAC ATA GCT 1200 Leu Leu Asp Asn Thr Ile Tyr Thr Gln Asn Glu Gly Phe Asn Ile Ala 385 390 395 400 AGT AAA AAT CTC AAA ACG GAA TTT AAT GGT CAG AAT AAG GCG GTA AAT 1248 Ser Lys Asn Leu Lys Thr Glu Phe Asn Gly Gln Asn Lys Ala Val Asn 405 410 415 AAA GAG GCT TAT GAA GAA ATC AGC CTA GAA CAT CTC GTT ATA TAT AGA 1296 Lys Glu Ala Tyr Glu Glu Ile Ser Leu Glu His Leu Val Ile Tyr Arg 420 425 430 ATA GCA ATG TGC AAG CCT GTA ATG TAC AAA AAT ACC GGT AAA TCT GAA 1344 Ile Ala Met Cys Lys Pro Val Met Tyr Lys Asn Thr Gly Lys Ser Glu 435 440 445 CAG TGT ATT ATT GTT AAT AAT GAG GAT TTA TTT TTC ATA GCT AAT AAA 1392 Gln Cys Ile Ile Val Asn Asn Glu Asp Leu Phe Phe Ile Ala Asn Lys 450 455 460 GAT AGT TTT TCA AAA GAT TTA GCT AAA GCA GAA ACT ATA GCA TAT AAT 1440 Asp Ser Phe Ser Lys Asp Leu Ala Lys Ala Glu Thr Ile Ala Tyr Asn 465 470 475 480 ACA CAA AAT AAT ACT ATA GAA AAT AAT TTT TCT ATA GAT CAG TTG ATT 1488 Thr Gln Asn Asn Thr Ile Glu Asn Asn Phe Ser Ile Asp Gln Leu Ile 485 490 495 TTA GAT AAT GAT TTA AGC AGT GGC ATA GAC TTA CCA AAT GAA AAC ACA 1536 Leu Asp Asn Asp Leu Ser Ser Gly Ile Asp Leu Pro Asn Glu Asn Thr 500 505 510 GAA CCA TTT ACA AAT TTT GAC GAC ATA GAT ATC CCT GTG TAT ATT AAA 1584 Glu Pro Phe Thr Asn Phe Asp Asp Ile Asp Ile Pro Val Tyr Ile Lys 515 520 525 CAA TCT GCT TTA AAA AAA ATT TTT GTG GAT GGA GAT AGC CTT TTT GAA 1632 Gln Ser Ala Leu Lys Lys Ile Phe Val Asp Gly Asp Ser Leu Phe Glu 530 535 540 TAT TTA CAT GCT CAA ACA TTT CCT TCT AAT ATA GAA AAT CTA CAA CTA 1680 Tyr Leu His Ala Gln Thr Phe Pro Ser Asn Ile Glu Asn Leu Gln Leu 545 550 555 560 ACG AAT TCA TTA AAT GAT GCT TTA AGA AAT AAT AAT AAA GTC TAT ACT 1728 Thr Asn Ser Leu Asn Asp Ala Leu Arg Asn Asn Asn Lys Val Tyr Thr 565 570 575 TTT TTT TCT ACA AAC CTT GTT GAA AAA GCT AAT ACA GTT GTA GGT GCT 1776 Phe Phe Ser Thr Asn Leu Val Glu Lys Ala Asn Thr Val Val Gly Ala 580 585 590 TCA CTT TTT GTA AAC TGG GTA AAA GGA GTA ATA GAT GAT TTT ACA TCT 1824 Ser Leu Phe Val Asn Trp Val Lys Gly Val Ile Asp Asp Phe Thr Ser 595 600 605 GAA TCC ACA CAA AAA AGT ACT ATA GAT AAA GTT TCA GAT GTA TCC ATA 1872 Glu Ser Thr Gln Lys Ser Thr Ile Asp Lys Val Ser Asp Val Ser Ile 610 615 620 ATT ATT CCC TAT ATA GGA CCT GCT TTG AAT GTA GGA AAT GAA ACA GCT 1920 Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn Val Gly Asn Glu Thr Ala 625 630 635 640 AAA GAA AAT TTT AAA AAT GCT TTT GAA ATA GGT GGA GCC GCT ATC TTA 1968 Lys Glu Asn Phe Lys Asn Ala Phe Glu Ile Gly Gly Ala Ala Ile Leu 645 650 655 ATG GAG TTT ATT CCA GAA CTT ATT GTA CCT ATA GTT GGA TTT TTT ACA 2016 Met Glu Phe Ile Pro Glu Leu Ile Val Pro Ile Val Gly Phe Phe Thr 660 665 670 TTA GAA TCA TAT GTA GGA AAT AAA GGG CAT ATT ATT ATG ACG ATA TCC 2064 Leu Glu Ser Tyr Val Gly Asn Lys Gly His Ile Ile Met Thr Ile Ser 675 680 685 AAT GCT TTA AAG AAA AGG GAT CAA AAA TGG ACA GAT ATG TAT GGT TTG 2112 Asn Ala Leu Lys Lys Arg Asp Gln Lys Trp Thr Asp Met Tyr Gly Leu 690 695 700 ATA GTA TCG CAG TGG CTC TCA ACG GTT AAT ACT CAA TTT TAT ACA ATA 2160 Ile Val Ser Gln Trp Leu Ser Thr Val Asn Thr Gln Phe Tyr Thr Ile 705 710 715 720 AAA GAA AGA ATG TAC AAT GCT TTA AAT AAT CAA TCA CAA GCA ATA GAA 2208 Lys Glu Arg Met Tyr Asn Ala Leu Asn Asn Gln Ser Gln Ala Ile Glu 725 730 735 AAA ATA ATA GAA GAT CAA TAT AAT AGA TAT AGT GAA GAA GAT AAA ATG 2256 Lys Ile Ile Glu Asp Gln Tyr Asn Arg Tyr Ser Glu Glu Asp Lys Met 740 745 750 AAT ATT AAC ATT GAT TTT AAT GAT ATA GAT TTT AAA CTT AAT CAA AGT 2304 Asn Ile Asn Ile Asp Phe Asn Asp Ile Asp Phe Lys Leu Asn Gln Ser 755 760 765 ATA AAT TTA GCA ATA AAC AAT ATA GAT GAT TTT ATA AAC CAA TGT TCT 2352 Ile Asn Leu Ala Ile Asn Asn Ile Asp Asp Phe Ile Asn Gln Cys Ser 770 775 780 ATA TCA TAT CTA ATG AAT AGA ATG ATT CCA TTA GCT GTA AAA AAG TTA 2400 Ile Ser Tyr Leu Met Asn Arg Met Ile Pro Leu Ala Val Lys Lys Leu 785 790 795 800 AAA GAC TTT GAT GAT AAT CTT AAG AGA GAT TTA TTG GAG TAT ATA GAT 2448 Lys Asp Phe Asp Asp Asn Leu Lys Arg Asp Leu Leu Glu Tyr Ile Asp 805 810 815 ACA AAT GAA CTA TAT TTA CTT GAT GAA GTA AAT ATT CTA AAA TCA AAA 2496 Thr Asn Glu Leu Tyr Leu Leu Asp Glu Val Asn Ile Leu Lys Ser Lys 820 825 830 GTA AAT AGA CAC CTA AAA GAC AGT ATA CCA TTT GAT CTT TCA CTA TAT 2544 Val Asn Arg His Leu Lys Asp Ser Ile Pro Phe Asp Leu Ser Leu Tyr 835 840 845 ACC AAG GAC ACA ATT TTA ATA CAA GTT TTT AAT AAT TAT ATT AGT AAT 2592 Thr Lys Asp Thr Ile Leu Ile Gln Val Phe Asn Asn Tyr Ile Ser Asn 850 855 860 ATT AGT AGT AAT GCT ATT TTA AGT TTA AGT TAT AGA GGT GGG CGT TTA 2640 Ile Ser Ser Asn Ala Ile Leu Ser Leu Ser Tyr Arg Gly Gly Arg Leu 865 870 875 880 ATA GAT TCA TCT GGA TAT GGT GCA ACT ATG AAT GTA GGT TCA GAT GTT 2688 Ile Asp Ser Ser Gly Tyr Gly Ala Thr Met Asn Val Gly Ser Asp Val 885 890 895 ATC TTT AAT GAT ATA GGA AAT GGT CAA TTT AAA TTA AAT AAT TCT GAA 2736 Ile Phe Asn Asp Ile Gly Asn Gly Gln Phe Lys Leu Asn Asn Ser Glu 900 905 910 AAT AGT AAT ATT ACG GCA CAT CAA AGT AAA TTC GTT GTA TAT GAT AGT 2784 Asn Ser Asn Ile Thr Ala His Gln Ser Lys Phe Val Val Tyr Asp Ser 915 920 925 ATG TTT GAT AAT TTT AGC ATT AAC TTT TGG GTA AGG ACT CCT AAA TAT 2832 Met Phe Asp Asn Phe Ser Ile Asn Phe Trp Val Arg Thr Pro Lys Tyr 930 935 940 AAT AAT AAT GAT ATA CAA ACT TAT CTT CAA AAT GAG TAT ACA ATA ATT 2880 Asn Asn Asn Asp Ile Gln Thr Tyr Leu Gln Asn Glu Tyr Thr Ile Ile 945 950 955 960 AGT TGT ATA AAA AAT GAC TCA GGA TGG AAA GTA TCT ATT AAG GGA AAT 2928 Ser Cys Ile Lys Asn Asp Ser Gly Trp Lys Val Ser Ile Lys Gly Asn 965 970 975 AGA ATA ATA TGG ACA TTA ATA GAT GTT AAT GCA AAA TCT AAA TCA ATA 2976 Arg Ile Ile Trp Thr Leu Ile Asp Val Asn Ala Lys Ser Lys Ser Ile 980 985 990 TTT TTC GAA TAT AGT ATA AAA GAT AAT ATA TCA GAT TAT ATA AAT AAA 3024 Phe Phe Glu Tyr Ser Ile Lys Asp Asn Ile Ser Asp Tyr Ile Asn Lys 995 1000 1005 TGG TTT TCC ATA ACT ATT ACT AAT GAT AGA TTA GGT AAC GCA AAT ATT 3072 Trp Phe Ser Ile Thr Ile Thr Asn Asp Arg Leu Gly Asn Ala Asn Ile 1010 1015 1020 TAT ATA AAT GGA AGT TTG AAA AAA AGT GAA AAA ATT TTA AAC TTA GAT 3120 Tyr Ile Asn Gly Ser Leu Lys Lys Ser Glu Lys Ile Leu Asn Leu Asp 1025 1030 1035 1040 AGA ATT AAT TCT AGT AAT GAT ATA GAC TTC AAA TTA ATT AAT TGT ACA 3168 Arg Ile Asn Ser Ser Asn Asp Ile Asp Phe Lys Leu Ile Asn Cys Thr 1045 1050 1055 GAT ACT ACT AAA TTT GTT TGG ATT AAG GAT TTT AAT ATT TTT GGT AGA 3216 Asp Thr Thr Lys Phe Val Trp Ile Lys Asp Phe Asn Ile Phe Gly Arg 1060 1065 1070 GAA TTA AAT GCT ACA GAA GTA TCT TCA CTA TAT TGG ATT CAA TCA TCT 3264 Glu Leu Asn Ala Thr Glu Val Ser Ser Leu Tyr Trp Ile Gln Ser Ser 1075 1080 1085 ACA AAT ACT TTA AAA GAT TTT TGG GGG AAT CCT TTA AGA TAC GAT ACA 3312 Thr Asn Thr Leu Lys Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr 1090 1095 1100 CAA TAC TAT CTG TTT AAT CAA GGT ATG CAA AAT ATC TAT ATA AAG TAT 3360 Gln Tyr Tyr Leu Phe Asn Gln Gly Met Gln Asn Ile Tyr Ile Lys Tyr 1105 1110 1115 1120 TTT AGT AAA GCT TCT ATG GGG GAA ACT GCA CCA CGT ACA AAC TTT AAT 3408 Phe Ser Lys Ala Ser Met Gly Glu Thr Ala Pro Arg Thr Asn Phe Asn 1125 1130 1135 AAT GCA GCA ATA AAT TAT CAA AAT TTA TAT CTT GGT TTA CGA TTT ATT 3456 Asn Ala Ala Ile Asn Tyr Gln Asn Leu Tyr Leu Gly Leu Arg Phe Ile 1140 1145 1150 ATA AAA AAA GCA TCA AAT TCT CGG AAT ATA AAT AAT GAT AAT ATA GTC 3504 Ile Lys Lys Ala Ser Asn Ser Arg Asn Ile Asn Asn Asp Asn Ile Val 1155 1160 1165 AGA GAA GGA GAT TAT ATA TAT CTT AAT ATT GAT AAT ATT TCT GAT GAA 3552 Arg Glu Gly Asp Tyr Ile Tyr Leu Asn Ile Asp Asn Ile Ser Asp Glu 1170 1175 1180 TCT TAC AGA GTA TAT GTT TTG GTG AAT TCT AAA GAA ATT CAA ACT CAA 3600 Ser Tyr Arg Val Tyr Val Leu Val Asn Ser Lys Glu Ile Gln Thr Gln 1185 1190 1195 1200 TTA TTT TTA GCA CCC ATA AAT GAT GAT CCT ACG TTC TAT GAT GTA CTA 3648 Leu Phe Leu Ala Pro Ile Asn Asp Asp Pro Thr Phe Tyr Asp Val Leu 1205 1210 1215 CAA ATA AAA AAA TAT TAT GAA AAA ACA ACA TAT AAT TGT CAG ATA CTT 3696 Gln Ile Lys Lys Tyr Tyr Glu Lys Thr Thr Tyr Asn Cys Gln Ile Leu 1220 1225 1230 TGC GAA AAA GAT ACT AAA ACA TTT GGG CTG TTT GGA ATT GGT AAA TTT 3744 Cys Glu Lys Asp Thr Lys Thr Phe Gly Leu Phe Gly Ile Gly Lys Phe 1235 1240 1245 GTT AAA GAT TAT GGA TAT GTT TGG GAT ACC TAT GAT AAT TAT TTT TGC 3792 Val Lys Asp Tyr Gly Tyr Val Trp Asp Thr Tyr Asp Asn Tyr Phe Cys 1250 1255 1260 ATA AGT CAG TGG TAT CTC AGA AGA ATA TCT GAA AAT ATA AAT AAA TTA 3840 Ile Ser Gln Trp Tyr Leu Arg Arg Ile Ser Glu Asn Ile Asn Lys Leu 1265 1270 1275 1280 AGG TTG GGA TGT AAT TGG CAA TTC ATT CCC GTG GAT GAA GGA TGG ACA 3888 Arg Leu Gly Cys Asn Trp Gln Phe Ile Pro Val Asp Glu Gly Trp Thr 1285 1290 1295 GAA TAA 3894 Glu

77 Met Pro Val Asn Ile Lys Asn Phe Asn Tyr Asn Asp Pro Ile Asn Asn 1 5 10 15 Asp Asp Ile Ile Met Met Glu Pro Phe Asn Asp Pro Gly Pro Gly Thr 20 25 30 Tyr Tyr Lys Ala Phe Arg Ile Ile Asp Arg Ile Trp Ile Val Pro Glu 35 40 45 Arg Phe Thr Tyr Gly Phe Gln Pro Asp Gln Phe Asn Ala Ser Thr Gly 50 55 60 Val Phe Ser Lys Asp Val Tyr Glu Tyr Tyr Asp Pro Thr Tyr Leu Lys 65 70 75 80 Thr Asp Ala Glu Lys Asp Lys Phe Leu Lys Thr Met Ile Lys Leu Phe 85 90 95 Asn Arg Ile Asn Ser Lys Pro Ser Gly Gln Arg Leu Leu Asp Met Ile 100 105 110 Val Asp Ala Ile Pro Tyr Leu Gly Asn Ala Ser Thr Pro Pro Asp Lys 115 120 125 Phe Ala Ala Asn Val Ala Asn Val Ser Ile Asn Lys Lys Ile Ile Gln 130 135 140 Pro Gly Ala Glu Asp Gln Ile Lys Gly Leu Met Thr Asn Leu Ile Ile 145 150 155 160 Phe Gly Pro Gly Pro Val Leu Ser Asp Asn Phe Thr Asp Ser Met Ile 165 170 175 Met Asn Gly His Ser Pro Ile Ser Glu Gly Phe Gly Ala Arg Met Met 180 185 190 Ile Arg Phe Cys Pro Ser Cys Leu Asn Val Phe Asn Asn Val Gln Glu 195 200 205 Asn Lys Asp Thr Ser Ile Phe Ser Arg Arg Ala Tyr Phe Ala Asp Pro 210 215 220 Ala Leu Thr Leu Met His Glu Leu Ile His Val Leu His Gly Leu Tyr 225 230 235 240 Gly Ile Lys Ile Ser Asn Leu Pro Ile Thr Pro Asn Thr Lys Glu Phe 245 250 255 Phe Met Gln His Ser Asp Pro Val Gln Ala Glu Glu Leu Tyr Thr Phe 260 265 270 Gly Gly His Asp Pro Ser Val Ile Ser Pro Ser Thr Asp Met Asn Ile 275 280 285 Tyr Asn Lys Ala Leu Gln Asn Phe Gln Asp Ile Ala Asn Arg Leu Asn 290 295 300 Ile Val Ser Ser Ala Gln Gly Ser Gly Ile Asp Ile Ser Leu Tyr Lys 305 310 315 320 Gln Ile Tyr Lys Asn Lys Tyr Asp Phe Val Glu Asp Pro Asn Gly Lys 325 330 335 Tyr Ser Val Asp Lys Asp Lys Phe Asp Lys Leu Tyr Lys Ala Leu Met 340 345 350 Phe Gly Phe Thr Glu Thr Asn Leu Ala Gly Glu Tyr Gly Ile Lys Thr 355 360 365 Arg Tyr Ser Tyr Phe Ser Glu Tyr Leu Pro Pro Ile Lys Thr Glu Lys 370 375 380 Leu Leu Asp Asn Thr Ile Tyr Thr Gln Asn Glu Gly Phe Asn Ile Ala 385 390 395 400 Ser Lys Asn Leu Lys Thr Glu Phe Asn Gly Gln Asn Lys Ala Val Asn 405 410 415 Lys Glu Ala Tyr Glu Glu Ile Ser Leu Glu His Leu Val Ile Tyr Arg 420 425 430 Ile Ala Met Cys Lys Pro Val Met Tyr Lys Asn Thr Gly Lys Ser Glu 435 440 445 Gln Cys Ile Ile Val Asn Asn Glu Asp Leu Phe Phe Ile Ala Asn Lys 450 455 460 Asp Ser Phe Ser Lys Asp Leu Ala Lys Ala Glu Thr Ile Ala Tyr Asn 465 470 475 480 Thr Gln Asn Asn Thr Ile Glu Asn Asn Phe Ser Ile Asp Gln Leu Ile 485 490 495 Leu Asp Asn Asp Leu Ser Ser Gly Ile Asp Leu Pro Asn Glu Asn Thr 500 505 510 Glu Pro Phe Thr Asn Phe Asp Asp Ile Asp Ile Pro Val Tyr Ile Lys 515 520 525 Gln Ser Ala Leu Lys Lys Ile Phe Val Asp Gly Asp Ser Leu Phe Glu 530 535 540 Tyr Leu His Ala Gln Thr Phe Pro Ser Asn Ile Glu Asn Leu Gln Leu 545 550 555 560 Thr Asn Ser Leu Asn Asp Ala Leu Arg Asn Asn Asn Lys Val Tyr Thr 565 570 575 Phe Phe Ser Thr Asn Leu Val Glu Lys Ala Asn Thr Val Val Gly Ala 580 585 590 Ser Leu Phe Val Asn Trp Val Lys Gly Val Ile Asp Asp Phe Thr Ser 595 600 605 Glu Ser Thr Gln Lys Ser Thr Ile Asp Lys Val Ser Asp Val Ser Ile 610 615 620 Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn Val Gly Asn Glu Thr Ala 625 630 635 640 Lys Glu Asn Phe Lys Asn Ala Phe Glu Ile Gly Gly Ala Ala Ile Leu 645 650 655 Met Glu Phe Ile Pro Glu Leu Ile Val Pro Ile Val Gly Phe Phe Thr 660 665 670 Leu Glu Ser Tyr Val Gly Asn Lys Gly His Ile Ile Met Thr Ile Ser 675 680 685 Asn Ala Leu Lys Lys Arg Asp Gln Lys Trp Thr Asp Met Tyr Gly Leu 690 695 700 Ile Val Ser Gln Trp Leu Ser Thr Val Asn Thr Gln Phe Tyr Thr Ile 705 710 715 720 Lys Glu Arg Met Tyr Asn Ala Leu Asn Asn Gln Ser Gln Ala Ile Glu 725 730 735 Lys Ile Ile Glu Asp Gln Tyr Asn Arg Tyr Ser Glu Glu Asp Lys Met 740 745 750 Asn Ile Asn Ile Asp Phe Asn Asp Ile Asp Phe Lys Leu Asn Gln Ser 755 760 765 Ile Asn Leu Ala Ile Asn Asn Ile Asp Asp Phe Ile Asn Gln Cys Ser 770 775 780 Ile Ser Tyr Leu Met Asn Arg Met Ile Pro Leu Ala Val Lys Lys Leu 785 790 795 800 Lys Asp Phe Asp Asp Asn Leu Lys Arg Asp Leu Leu Glu Tyr Ile Asp 805 810 815 Thr Asn Glu Leu Tyr Leu Leu Asp Glu Val Asn Ile Leu Lys Ser Lys 820 825 830 Val Asn Arg His Leu Lys Asp Ser Ile Pro Phe Asp Leu Ser Leu Tyr 835 840 845 Thr Lys Asp Thr Ile Leu Ile Gln Val Phe Asn Asn Tyr Ile Ser Asn 850 855 860 Ile Ser Ser Asn Ala Ile Leu Ser Leu Ser Tyr Arg Gly Gly Arg Leu 865 870 875 880 Ile Asp Ser Ser Gly Tyr Gly Ala Thr Met Asn Val Gly Ser Asp Val 885 890 895 Ile Phe Asn Asp Ile Gly Asn Gly Gln Phe Lys Leu Asn Asn Ser Glu 900 905 910 Asn Ser Asn Ile Thr Ala His Gln Ser Lys Phe Val Val Tyr Asp Ser 915 920 925 Met Phe Asp Asn Phe Ser Ile Asn Phe Trp Val Arg Thr Pro Lys Tyr 930 935 940 Asn Asn Asn Asp Ile Gln Thr Tyr Leu Gln Asn Glu Tyr Thr Ile Ile 945 950 955 960 Ser Cys Ile Lys Asn Asp Ser Gly Trp Lys Val Ser Ile Lys Gly Asn 965 970 975 Arg Ile Ile Trp Thr Leu Ile Asp Val Asn Ala Lys Ser Lys Ser Ile 980 985 990 Phe Phe Glu Tyr Ser Ile Lys Asp Asn Ile Ser Asp Tyr Ile Asn Lys 995 1000 1005 Trp Phe Ser Ile Thr Ile Thr Asn Asp Arg Leu Gly Asn Ala Asn Ile 1010 1015 1020 Tyr Ile Asn Gly Ser Leu Lys Lys Ser Glu Lys Ile Leu Asn Leu Asp 1025 1030 1035 1040 Arg Ile Asn Ser Ser Asn Asp Ile Asp Phe Lys Leu Ile Asn Cys Thr 1045 1050 1055 Asp Thr Thr Lys Phe Val Trp Ile Lys Asp Phe Asn Ile Phe Gly Arg 1060 1065 1070 Glu Leu Asn Ala Thr Glu Val Ser Ser Leu Tyr Trp Ile Gln Ser Ser 1075 1080 1085 Thr Asn Thr Leu Lys Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr 1090 1095 1100 Gln Tyr Tyr Leu Phe Asn Gln Gly Met Gln Asn Ile Tyr Ile Lys Tyr 1105 1110 1115 1120 Phe Ser Lys Ala Ser Met Gly Glu Thr Ala Pro Arg Thr Asn Phe Asn 1125 1130 1135 Asn Ala Ala Ile Asn Tyr Gln Asn Leu Tyr Leu Gly Leu Arg Phe Ile 1140 1145 1150 Ile Lys Lys Ala Ser Asn Ser Arg Asn Ile Asn Asn Asp Asn Ile Val 1155 1160 1165 Arg Glu Gly Asp Tyr Ile Tyr Leu Asn Ile Asp Asn Ile Ser Asp Glu 1170 1175 1180 Ser Tyr Arg Val Tyr Val Leu Val Asn Ser Lys Glu Ile Gln Thr Gln 1185 1190 1195 1200 Leu Phe Leu Ala Pro Ile Asn Asp Asp Pro Thr Phe Tyr Asp Val Leu 1205 1210 1215 Gln Ile Lys Lys Tyr Tyr Glu Lys Thr Thr Tyr Asn Cys Gln Ile Leu 1220 1225 1230 Cys Glu Lys Asp Thr Lys Thr Phe Gly Leu Phe Gly Ile Gly Lys Phe 1235 1240 1245 Val Lys Asp Tyr Gly Tyr Val Trp Asp Thr Tyr Asp Asn Tyr Phe Cys 1250 1255 1260 Ile Ser Gln Trp Tyr Leu Arg Arg Ile Ser Glu Asn Ile Asn Lys Leu 1265 1270 1275 1280 Arg Leu Gly Cys Asn Trp Gln Phe Ile Pro Val Asp Glu Gly Trp Thr 1285 1290 1295 Glu

78 AGATCTCGAT CCCGCGAAAT TAATACGACT CACTATAGGG GAATTGTGAG CGGATAACAA 60 TTCCCCTCTA GAAATAATTT TGTTTAACTT TAAGAAGGAG ATATACC ATG GGC CAT 116 Met Gly His 1 CAT CAT CAT CAT CAT CAT CAT CAT CAC AGC AGC GGC CAT ATC GAA GGT 164 His His His His His His His His His Ser Ser Gly His Ile Glu Gly 5 10 15 CGT CAT ATG GCT AGC ATG GCT GAC ACA ATT TTA ATA CAA GTT TTT AAT 212 Arg His Met Ala Ser Met Ala Asp Thr Ile Leu Ile Gln Val Phe Asn 20 25 30 35 AAT TAT ATT AGT AAT ATT AGT AGT AAT GCT ATT TTA AGT TTA AGT TAT 260 Asn Tyr Ile Ser Asn Ile Ser Ser Asn Ala Ile Leu Ser Leu Ser Tyr 40 45 50 AGA GGT GGG CGT TTA ATA GAT TCA TCT GGA TAT GGT GCA ACT ATG AAT 308 Arg Gly Gly Arg Leu Ile Asp Ser Ser Gly Tyr Gly Ala Thr Met Asn 55 60 65 GTA GGT TCA GAT GTT ATC TTT AAT GAT ATA GGA AAT GGT CAA TTT AAA 356 Val Gly Ser Asp Val Ile Phe Asn Asp Ile Gly Asn Gly Gln Phe Lys 70 75 80 TTA AAT AAT TCT GAA AAT AGT AAT ATT ACG GCA CAT CAA AGT AAA TTC 404 Leu Asn Asn Ser Glu Asn Ser Asn Ile Thr Ala His Gln Ser Lys Phe 85 90 95 GTT GTA TAT GAT AGT ATG TTT GAT AAT TTT AGC ATT AAC TTT TGG GTA 452 Val Val Tyr Asp Ser Met Phe Asp Asn Phe Ser Ile Asn Phe Trp Val 100 105 110 115 AGG ACT CCT AAA TAT AAT AAT AAT GAT ATA CAA ACT TAT CTT CAA AAT 500 Arg Thr Pro Lys Tyr Asn Asn Asn Asp Ile Gln Thr Tyr Leu Gln Asn 120 125 130 GAG TAT ACA ATA ATT AGT TGT ATA AAA AAT GAC TCA GGA TGG AAA GTA 548 Glu Tyr Thr Ile Ile Ser Cys Ile Lys Asn Asp Ser Gly Trp Lys Val 135 140 145 TCT ATT AAG GGA AAT AGA ATA ATA TGG ACA TTA ATA GAT GTT AAT GCA 596 Ser Ile Lys Gly Asn Arg Ile Ile Trp Thr Leu Ile Asp Val Asn Ala 150 155 160 AAA TCT AAA TCA ATA TTT TTC GAA TAT AGT ATA AAA GAT AAT ATA TCA 644 Lys Ser Lys Ser Ile Phe Phe Glu Tyr Ser Ile Lys Asp Asn Ile Ser 165 170 175 GAT TAT ATA AAT AAA TGG TTT TCC ATA ACT ATT ACT AAT GAT AGA TTA 692 Asp Tyr Ile Asn Lys Trp Phe Ser Ile Thr Ile Thr Asn Asp Arg Leu 180 185 190 195 GGT AAC GCA AAT ATT TAT ATA AAT GGA AGT TTG AAA AAA AGT GAA AAA 740 Gly Asn Ala Asn Ile Tyr Ile Asn Gly Ser Leu Lys Lys Ser Glu Lys 200 205 210 ATT TTA AAC TTA GAT AGA ATT AAT TCT AGT AAT GAT ATA GAC TTC AAA 788 Ile Leu Asn Leu Asp Arg Ile Asn Ser Ser Asn Asp Ile Asp Phe Lys 215 220 225 TTA ATT AAT TGT ACA GAT ACT ACT AAA TTT GTT TGG ATT AAG GAT TTT 836 Leu Ile Asn Cys Thr Asp Thr Thr Lys Phe Val Trp Ile Lys Asp Phe 230 235 240 AAT ATT TTT GGT AGA GAA TTA AAT GCT ACA GAA GTA TCT TCA CTA TAT 884 Asn Ile Phe Gly Arg Glu Leu Asn Ala Thr Glu Val Ser Ser Leu Tyr 245 250 255 TGG ATT CAA TCA TCT ACA AAT ACT TTA AAA GAT TTT TGG GGG AAT CCT 932 Trp Ile Gln Ser Ser Thr Asn Thr Leu Lys Asp Phe Trp Gly Asn Pro 260 265 270 275 TTA AGA TAC GAT ACA CAA TAC TAT CTG TTT AAT CAA GGT ATG CAA AAT 980 Leu Arg Tyr Asp Thr Gln Tyr Tyr Leu Phe Asn Gln Gly Met Gln Asn 280 285 290 ATC TAT ATA AAG TAT TTT AGT AAA GCT TCT ATG GGG GAA ACT GCA CCA 1028 Ile Tyr Ile Lys Tyr Phe Ser Lys Ala Ser Met Gly Glu Thr Ala Pro 295 300 305 CGT ACA AAC TTT AAT AAT GCA GCA ATA AAT TAT CAA AAT TTA TAT CTT 1076 Arg Thr Asn Phe Asn Asn Ala Ala Ile Asn Tyr Gln Asn Leu Tyr Leu 310 315 320 GGT TTA CGA TTT ATT ATA AAA AAA GCA TCA AAT TCT CGG AAT ATA AAT 1124 Gly Leu Arg Phe Ile Ile Lys Lys Ala Ser Asn Ser Arg Asn Ile Asn 325 330 335 AAT GAT AAT ATA GTC AGA GAA GGA GAT TAT ATA TAT CTT AAT ATT GAT 1172 Asn Asp Asn Ile Val Arg Glu Gly Asp Tyr Ile Tyr Leu Asn Ile Asp 340 345 350 355 AAT ATT TCT GAT GAA TCT TAC AGA GTA TAT GTT TTG GTG AAT TCT AAA 1220 Asn Ile Ser Asp Glu Ser Tyr Arg Val Tyr Val Leu Val Asn Ser Lys 360 365 370 GAA ATT CAA ACT CAA TTA TTT TTA GCA CCC ATA AAT GAT GAT CCT ACG 1268 Glu Ile Gln Thr Gln Leu Phe Leu Ala Pro Ile Asn Asp Asp Pro Thr 375 380 385 TTC TAT GAT GTA CTA CAA ATA AAA AAA TAT TAT GAA AAA ACA ACA TAT 1316 Phe Tyr Asp Val Leu Gln Ile Lys Lys Tyr Tyr Glu Lys Thr Thr Tyr 390 395 400 AAT TGT CAG ATA CTT TGC GAA AAA GAT ACT AAA ACA TTT GGG CTG TTT 1364 Asn Cys Gln Ile Leu Cys Glu Lys Asp Thr Lys Thr Phe Gly Leu Phe 405 410 415 GGA ATT GGT AAA TTT GTT AAA GAT TAT GGA TAT GTT TGG GAT ACC TAT 1412 Gly Ile Gly Lys Phe Val Lys Asp Tyr Gly Tyr Val Trp Asp Thr Tyr 420 425 430 435 GAT AAT TAT TTT TGC ATA AGT CAG TGG TAT CTC AGA AGA ATA TCT GAA 1460 Asp Asn Tyr Phe Cys Ile Ser Gln Trp Tyr Leu Arg Arg Ile Ser Glu 440 445 450 AAT ATA AAT AAA TTA AGG TTG GGA TGT AAT TGG CAA TTC ATT CCC GTG 1508 Asn Ile Asn Lys Leu Arg Leu Gly Cys Asn Trp Gln Phe Ile Pro Val 455 460 465 GAT GAA GGA TGG ACA GAA TAACTCGAG 1535 Asp Glu Gly Trp Thr Glu 470

79 Met Gly His His His His His His His His His His Ser Ser Gly His 1 5 10 15 Ile Glu Gly Arg His Met Ala Ser Met Ala Asp Thr Ile Leu Ile Gln 20 25 30 Val Phe Asn Asn Tyr Ile Ser Asn Ile Ser Ser Asn Ala Ile Leu Ser 35 40 45 Leu Ser Tyr Arg Gly Gly Arg Leu Ile Asp Ser Ser Gly Tyr Gly Ala 50 55 60 Thr Met Asn Val Gly Ser Asp Val Ile Phe Asn Asp Ile Gly Asn Gly 65 70 75 80 Gln Phe Lys Leu Asn Asn Ser Glu Asn Ser Asn Ile Thr Ala His Gln 85 90 95 Ser Lys Phe Val Val Tyr Asp Ser Met Phe Asp Asn Phe Ser Ile Asn 100 105 110 Phe Trp Val Arg Thr Pro Lys Tyr Asn Asn Asn Asp Ile Gln Thr Tyr 115 120 125 Leu Gln Asn Glu Tyr Thr Ile Ile Ser Cys Ile Lys Asn Asp Ser Gly 130 135 140 Trp Lys Val Ser Ile Lys Gly Asn Arg Ile Ile Trp Thr Leu Ile Asp 145 150 155 160 Val Asn Ala Lys Ser Lys Ser Ile Phe Phe Glu Tyr Ser Ile Lys Asp 165 170 175 Asn Ile Ser Asp Tyr Ile Asn Lys Trp Phe Ser Ile Thr Ile Thr Asn 180 185 190 Asp Arg Leu Gly Asn Ala Asn Ile Tyr Ile Asn Gly Ser Leu Lys Lys 195 200 205 Ser Glu Lys Ile Leu Asn Leu Asp Arg Ile Asn Ser Ser Asn Asp Ile 210 215 220 Asp Phe Lys Leu Ile Asn Cys Thr Asp Thr Thr Lys Phe Val Trp Ile 225 230 235 240 Lys Asp Phe Asn Ile Phe Gly Arg Glu Leu Asn Ala Thr Glu Val Ser 245 250 255 Ser Leu Tyr Trp Ile Gln Ser Ser Thr Asn Thr Leu Lys Asp Phe Trp 260 265 270 Gly Asn Pro Leu Arg Tyr Asp Thr Gln Tyr Tyr Leu Phe Asn Gln Gly 275 280 285 Met Gln Asn Ile Tyr Ile Lys Tyr Phe Ser Lys Ala Ser Met Gly Glu 290 295 300 Thr Ala Pro Arg Thr Asn Phe Asn Asn Ala Ala Ile Asn Tyr Gln Asn 305 310 315 320 Leu Tyr Leu Gly Leu Arg Phe Ile Ile Lys Lys Ala Ser Asn Ser Arg 325 330 335 Asn Ile Asn Asn Asp Asn Ile Val Arg Glu Gly Asp Tyr Ile Tyr Leu 340 345 350 Asn Ile Asp Asn Ile Ser Asp Glu Ser Tyr Arg Val Tyr Val Leu Val 355 360 365 Asn Ser Lys Glu Ile Gln Thr Gln Leu Phe Leu Ala Pro Ile Asn Asp 370 375 380 Asp Pro Thr Phe Tyr Asp Val Leu Gln Ile Lys Lys Tyr Tyr Glu Lys 385 390 395 400 Thr Thr Tyr Asn Cys Gln Ile Leu Cys Glu Lys Asp Thr Lys Thr Phe 405 410 415 Gly Leu Phe Gly Ile Gly Lys Phe Val Lys Asp Tyr Gly Tyr Val Trp 420 425 430 Asp Thr Tyr Asp Asn Tyr Phe Cys Ile Ser Gln Trp Tyr Leu Arg Arg 435 440 445 Ile Ser Glu Asn Ile Asn Lys Leu Arg Leu Gly Cys Asn Trp Gln Phe 450 455 460 Ile Pro Val Asp Glu Gly Trp Thr Glu 465 470

80 CGCCATGGCT GACACAATTT TAATACAAGT 30

81 GCCTCGAGTT ATTCTGTCCA TCCTTCATCC AC 32

/note= “The asparagine residue at this position contains an amide group.”

82 Cys Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn 1 5 10