CA1341491C

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Publication number: CA1341491C
Application number: CA000455960A
Authority: CA
Inventors: James Mon Lee; Axel Ullrich
Original assignee: Genentech Inc
Current assignee: Genentech Inc
Priority date: 1983-06-20
Filing date: 1984-06-06
Publication date: 2005-12-20
Anticipated expiration: 2022-12-20

Abstract

Human insulin-like growth factor (IGF) and epidermal growth factor (EGF) have been successfully prepared via recombinant DNA technology as mature products of expression, processing and secretion from a host culture.

Description

Figure d shows a sequence of D~IA and deduced snort fusion protein containing IGF-I.
Figure 9 depicts a plasmid used in the present construction.
rigure 10 shows the DNA and protein sequence of IGF-I fused with alpha factor pre-pro sequence.
f=igure 11 is a vector containing alpha factor promotor and pre-pro sequence fused to IGF-I.
Figure 12 sho:as the yeast invertase signal fused to IGF-I.
Figure 13 shows the parental plasmid containing the yeast PGK
promotor.
Figure 1~ depicts a yeast expression vector containing PGK
promotor, invertase signal and human IGF-I gene.
Figurz 15 is the synthetic rgi~A used to construct the coding sequence of mature human EGF.
Figure 16 depicts the yeast alaoa factor "pre-pro" sequence fused to the human EGF coding sequence.
Figure 17 depicts the yeast invertase signal sequence fused to the human EGF coding sequence.
Figure 18 shoes the coding sequence for human IGF-II.
Detailed Description A. Definitions As used herein, "human IGF" and "human EGF" denotes human i nsul i n-1 i ke growth factor and human epi dermal gro' th factor, produced by microbial or cell cultures systems and bioactive forms comprising the amino acid sequence corresponding to~i~uman IGF and human cGF othera~ise native to human tissue. The human IGF and EGF
proteins produced herein have been defined by means of DNA, gene, and deductive amino acid sequencing. It will be understood that inasmuch as natural allelic variations exist and occur from individual to individual, as demonstrated by (an) amino acid differences) in the overall sequence or by deletions, substitutions, insertions, inversions, or additions of one or more amino acids of said sequences, the present invention is intended to embrace of all such allelic variations of ~he t~.ro molecules involved. In addition, the location of and the degree of glycosylation depends upon the nature of the recombinant host organism employed and such variations as may occur are included :~ri thi n thz arnbi t of tiai s i ovsn ti an. F i nal ly, toe po ten ti al ~xi sts in the use of DNA technology for the preparation of various derivatives of human IGF and human EGF by simple modification of the underlying gene sequence for such molecules. Such modifications could be accompiisi~~d by means of site directed mutagenesis of the underlying DNA, as an example. All such modifications resulting in deri vati ves of human IGF and human EGF are i ncl uded Lei thi n the scope oP the present invention so long as the essential characteristic human IGF and human EGF activities remain unaffocied in kind.
"Essentially pure form" when used to describe the state of human IGF or human EGF produced by this invention mzans that the proteins are free of proteins or other materials normally associated with' human IGF or human EGF yvhen produced by non-recombinant cells, i.e.
in their "native" environments.
"Expression vector" includes vectors which are capable of expressing DNA sequences contained therein, where such sequences are operably linked to other sequences capable of effecting toeir expression, i.e., promotor/operator sequences. In sum, "expression vector" is given a functional definition: any DNA sequence which is capable of effecting expression of a sp~cifi~d DNA code disposed therein. In general, expression vectors of utility in recombinant DNA teconi ques are often i n the form of "pl asmi ds" whi;.h refer to circular double stranded DNA loops which in their vector form, are not bound to the chromosone. In the present specification, "plasmid" and "vector" are used interchangably as the plasmid is the most commonly used fow~ of vector. However, toe i nventi on i s intended to include such other fon;~s of expression vectors which function equivalently and which become known in the art subsequently.
"Recombinant host cells" refers to cells ;~hich have been transformed with such vectors. Thus, the human IGF and human EGF
rnol ecul es produced by such cel l s can be referred to as "recombi nant ~numan IGF" and "recomuinant human EGF".
B. Host Cell Cultures and Vectors The vec torn and ~netliods di scl osed herei n a re sui taol s f or use i n host cells over a wide range of prokaryotic and eukaryotic organisms.
In general, of course, prokaryotes are preferred for cloning of DtJA sequences in constructing the vectors useful in the invention.
For exampl e, E. col i K12 strai n 294 (ATCC PJo. 31440 i s particul arly useful. Other microbial strains w~ich may be used include _E. coli strains such as E. coli B, and E. coli X1775 (ATTC No. 31537). The aforementioned strains, as well as E. coli W3110 (F-, a-, prototrophic, ATTC No. 27325), bacilli such as Bacillus subtilus, and other enterobacteriaceae such as Salmonella typhimurium or Serratia marcesans, and various pseudomonas species may be used.
These examples are, of course, intended to be illustrative rather than limiting.
In general, plasmid vectors containing replicon and control sequences which are derived from species compatible v~itn the host cel l are used i n connection vi th these hosts. The vector ordi nari ly carries a replication site, as well as marking sequences which are capa~l2 of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR 322, a plasmid deri ved f rom an E . col i sj~ec i es ( Bol i var, et al . , Gene 2: 95 (1977)). pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. Tie pBR322 plasmid, or other microbial plasmid must also contain, or be modified to contain, promoters which can b~ used by the microbial organism for expression of its own proteins. Those promoters most commonly used in recombinant DNA construction include the ~-lactamase (penicillinase) and lactose promoter systems (Chang et al, Nature, 275: 015 (1978); Itakura, et al, Science, 198: 1050 (1977); (GOeddel, et al iJatur~ 281: 544 (1979)) and a tryptophan !+r~1 ~...n..~~+.~... j~ j~n.~ / j~n.~.lol~ .'~ al ~;',i~ln;.. I,ri.lj lo.~j ~. /~1,C7 r' i ~ i ( 1980 ) ; EP0 Appl Publ i~lo. 0030770) . Whi 1 a these a re the rnos t commonly used, other microbial promoters have been discovereJ and utilized, and details concerning their nucleotide sequences have been published, enabling a skilled worker to ligate then functionally with plasmid vectors (Siebenlist, et al, Cell 20: 2G9 (1980)).
In addition to prokaryates, eukaryotic microbes, such as yeast cultures may also be used. Saccharomyces cerevisiae, or common baker's yeast is the roost commonly used among eukaryotic microorganisms, although a number of other strains are comrnonly availa5le. For expression in Saccharomyces, the plasmid YRp7, fir example, (Stinchcomb, et al, Nature, 282: 39 (1979); Kingsman et al, Gene, 7: 141 (1979); Tschemper, et al, Gene, 10: 157 (1980)) is commonly used. This plasmid already contains the trill gene whic~
provides a selection marker for a mutant strain of yeast lacking the abi 1 i ty to grow i n tryp tophan, for exaopl a ATCC i~do. 44070 or PEP4-1 (Jones, Genetics, 85: 12 (1977)). The presence of the trill lesion as a characteristic of the yeast host cell genome then provides an 1~

effective environment for detecting transformation by grow th in the absence of tryptophan.
Suitable promoting sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase (Hitzeinan, et al., J. 8iol.
Chem., 255: 2073 (1980)) or other glycolytic enzymes (Hess, et al, J. Adv. Enzyme Reg., 7: 149 (1868); Holland, et al, Biochemistry, 17: 4900 (1978)), such as enolase, glyceralde~iyde-3-phosphate dehydrogenase, hexokinase; pyruvate decarboxylase, phosphofructokinase, glucose-5-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. In constructing suitable expression plasmids, the termination sequences associated ~vith these genes are also ligated into the expression vector 3' of the sequence desired to be expressed to provide polyadenyl ati on of the i;~RivA and ter;ni ~idtl Jrt. Otoea~ promoters, ,phi ch have the additional advantage of transcription controlled by growth condi ti ons are the promoter regi ons f or al coi~ol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolise, and the aforementioned glyceraldehyde-3-nhosuhate dehydrogenase, and enzymes responsible for maltose and 13 4149 ~
galactose utilization (Holland, ibid.). Any plasmid vector cootai ni ng yeast-compati bi a promoter, on gi n of repl i cati o;i and termination sequences is suitable.
In addition to microorganisms, cultures of cells derived from multicellular organisms may also be used as hosts. In principle, any such cel 1 cul lure i s wor~:abl e, :v he ther f rur~ verteorate or invertebrate culture. However interest has been greatest in vertebrate cells, and propogation of vertebrate cells in culture (tissue culture) has become a routine procedure in recent years [Tissue Culture, Academic Press, .Cruse and Patterson, editors (1973)x. Examples of such useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and W138, 3HK, COS-7 and MDCK cell lines. Expression vectors for such cells ordinarily ~5 include (if necessary) an origin of replication, a prornoter located in front o.f the gene to be expressed, along r~ith any nec~ssery ribosome binding sites, NNA splice sites, polyadenylation site, and transcriptional terminator sequences.
- For use i n mammal i an cel l s, the control functions on toe expression vectors are often provided by viral material. For example, coimnonly used promoters are derived from polyoma, Adenovirus 2, and most frequently Simian Virus ~0 (SV~O). The early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication (Fiers, et al, idature, 273: 113 ( 1978). ~ Smal l er or l arger SV4J fragments rnay also be used, provided there is included the approximately 250 by sequence extending from the Hind III site to»rard the 8g1 I site located in the viral origin of replication.
Further, it is also poS5lble, and often desirable, to utilize promoter or control sequences normally associated :rito toe desired gene sequence, provide such control sequences are compatible with tree host cell systems.

An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV:I~0 or other viral (e. g. Polyoma, Adeno, VSV, BPV, etc.) source, or may be provided by the frost cell chromosomal replication mechanism. If the vector is integrated into the host cell ci~romosome, the latter is often sufficient.
C. ~~ethods Employed If cells without formidable cell wall barriers are used as host cells, transfection is carried out by the calcium phosphate precipitation rnetihod as described by Graham and Van der Eb, Virolo y, 52: 545 (1878). However, other methods for introducing O~dA i nto cel 1 s such as by nucl ear i njecti on or by protopl ast fusi on may also be used.
If prokaryotic cells or cells which contain substantial cell wall constructions are used, the preferred method of transfection is calcium treatment using calcium ci~loride as described by Cohen, F.N.
et al Proc. iJatl. Aced. Sci. (USA), 58: 2110 (1972).
Construction of suitable vectors containing the desired coding and control sequences employ standard ligation techniques. Isolated plasmids or D~JA fragments are cleaved, tailored, and religated in the form desired to form the plasmids required.
Cleavage is performed by treating with restriction enzyme (or enyzmes) in suitable buffer. In general, about 1 wg plasmid or D?JA' fragments is used with about 1 unit of enzyme in about ZO ~l of buffer solution. (Aj~propriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer.) Incubation times of about 1 hour at 37°C are ~~rorkable. After incubations, protein is removed vy extraction vit~~ phenol and chloroform, and the nucleic acid is recovered from the aqueous fracti on by preci pi tati on ~r~i th ethanol .
.. . a 134t49t If blunt ends are required, the preparation is treated for 15 minutes at 15" ;vita 10 units of Polymerase I (Klenotv), phenol-chloroform extracted, and ethanol precipitated.
Size separation of the cleaved fragments is performed using 5 percent polyacrylamide gel described by Goeddel, D., et al, Nucleic Acids Res., 8: 4057 (1980) incorporated herein by reference.
For ligation approximately equimolar amounts of the desired components, suitably end tailored to provide correct matching are treated with about 10 units T4 DNA lipase per 0.5 ug DNA. (When cleaved vectors are used as components, it may be useful to prevent religation of the cleaved vector by pretreatment with bacterial alkaline phosphatase.) Far analysis to confirm correct sequences in plasmids constructed, the ligation mixtures are used to transform E. coli ~C12 strain 294 (ATCC 31440), and successful transfornants selected by ampicillin resistance where appropriate. Plasmids from the transfonnants are prepared, analyzed by restriction and/or sequenced by the method of P4essing, et al, Nucleic Acids Res., 9:309 (1981) or by the method of Maxam, et al, Methods in Enzymology, 65:499 (1980).
Examples ' The following examples are intended to illustrate but not to limit the present invention.
Synthesis and Expression of Human IGF-1 Enzymes were obtained from the follo;ving suppliers:
New England Biolabs: restriction enzymes, T4 DNA lipase Bethesda research Labs: restriction enzymes, Bact. Ali;aline ~~ 41491 Phos.
Boehringer-Mannheim: E. cola DNA Polymerase I (Klenow) p+L Biochemicals: Polynucleotide kinase, Terminal Nucleotidyl Transferase New England Nuclear:
pBR322 [oligo(dG)-tailed] DNA
Reagents:
BioRad: Bis Acrylamide, Acrylamide, TEMED
Sigma.: Ammonium Persulfate Amersham: 10210 7 32p ATP >5000 Ci/mmol; 10165 a 32p dCTP 400 >Ci/mmol.
Solutions and Media: 1X TBE:~ .54M Tris Base, 0.54 M Horic Acid, .017 M Na2 EDTA.
Difco: Yeast Nitrogenous Base (YNB); Tryptone, Yeast Extract: Bacto-Agar; Casamino Acids.
Autoradiography:
Kodak X-0 mat AR XAR-2~ Film Glass Beads:
0.45-0.50 mM B.Braun Melsungen AG
LB medium (per liter) lOg NaCl; 5g yeast extract: lOg tryptone; .17m1 NaOH (50 percent) LB Agar (per liter):
lOg tryptone; 5g yeast extract; .5g NaCl; 15g Bacto-Agar:
adjusted to pH 7.5 with NaOH.
Antibiotics:
Tetracycline (5 ~g/ml) in all mediums; Ampicillin (20 ~g/ml) .in all mediums (plates or liquid) ~3 4149 ii9 i~ledi um (per 1 i ter) 6g Na2HP04 (anhydrous); 1g NH4C1; 3g KH2P04; .5g NaCI ; 1 ;nit iigSO,~; 0.5 percent (svlv) gl ucose; 0.5 percent (:v/v) Casamino Acids; .0001 percent Thiamine-HCI.
YNB-CAA (per liter):
5.7g Yeast Nitrogenous Base (without Amino Acids); 10 mg adenine; 10 mg uracil; 5g Casamino Acids; 20g Glucose.
YiJ3-CAA agar pl ates ( per l i ter) Same as YN3-CAA + 30g agar.
Standard Ligation Conditions:
10-fold molar excess of insert (or linker) to vector. 1X T4 DNA ligase buffer and 400-800 U T4 DNA ligase; 14° - 12-16 ~~ours.
Standard Kination Conditions:
1X Polynucleotide Kinase buffer; 15 U polynucleotide kinase;
37° 00 minutes; followed by reaction termination by heating to 6~° for 10 minutes.
1X Kinase Buffer:
70 m~~1 Tris-HC1 (pH 7.6); 10 mid i~1gC12; 5 nil DTT
' 1X T4 DNA Ligase Buffer:
50 m~~l Tris-HCl (pH 7.8); 10 rust ~4gC12; 20 r~i~t DTT; 1 mt1 rATP.
Construction, Strategy and Selection of a DiJA Sequence.
The 1° protein structure of the human IGF-1 molecule has been determined (1). Based upon t~~is protein sequence and the genetic code, a DNA sequence coding for mature human IGF-1 protein, including all possible base substitutions at any one base position, was determined by computer analysis (Genentech Untrans Program).

1s 41491 Using a restriction sits analysis program (Genentech Asearch Program), all potential restriction sites located in all possible DNA sequences consistently coding for the same protein were found.
Three sites internal to the coding sequence were selected: PstI, BamHI, and AvaII. Two additional sites were placed at the ends, just outside of the coding sequence of the mature protein: one EcoRI site before the initiation codon, AUG, and the SaII site following the termination colon, TAG of the coding sequence. The choice of these sites facilitated the cloning of the coding sequencz in separate parts, each of which subsequently could be excised and then assembled to form an intact synthetic IGF-1 gene. This construction involved the assembly of 4 parts, 2 parts forming the left half, 2 parts fo ,~ing the right half. Each part consisted of two single strands of chemically synthesized DidA (see Fig. 1).
~5 Proposed synthetic fragments were also analyzed for internal complementarity.
The constructions used to generate these four parts employed the use of DNA Polymerase I repair synthesis of synthetic 20 oligonucleotide substrates having 9-10 by stretches of complementary sequence at their 3' termini. In the presence of DNA Polymerase I
(K1?now) and the four deoxynwleoside triphosphates, these primer-templates were extended to become full-length double-stranded DNAs. To prevent priming at locations other than the desired . 25 portions as well as self-hybridizations, each set of Single-stranded DNAs were analyzed by a computer program (Genentech Homology Program), and wherever possible, sequences which would have potentially led to hairpin loops, self-priming, or rnis-priming, were eliminated by alternate colon usage. Each of these four 30 double-stranded DidAs were synthesized to include 9-12 additional by of non-IGF-1 coding DNA at each end (see Fig. 2). This additional DNA ,~~as included to allow generation of sticky ends by restriction enzyme digestion. The sticky ends thus formed facilitated the ligation of the double-stranded pieces to contiguous coding sections 35 of the synthetic gene or into a cloning vehicle.

1~~14J1._ The 9-12 extra by of double stranded DNA beyond the restriction site at the end of each part (see Fig. 2) allo~red for the TdT-mediated formation of single-stranded oligodeoxycytidine strands at the 3' ends of each double-stranded DNA section. These oligodeoxycytidine tailed double-stranded DNAs could then be anneaiied into a complementary oligodeoxyguanosine tailed Pstl site of a cloning vehicle. Once cloned, and sequenced to ensure the correct base sequences, the parts could be easily isolated and 1 i gated f of 1 ovai ng restri cti on enzyme cl eavage at the res tri cti on sites selected at the ends of each of the four parts, to form the intact synthetic IGF-1 gene.
The method used successfully here was similar to that described by Rossi et al. (28); ho:~ever, attempts at the construction and cloning of the IGF-1 coding sequence using the Rossi et al. method (28) ~~ith,only two base pairs of extra DNA beyond the restriction enzyrne recognition sites repeatedly failed. The method employed here also differs from the Rossi et al. procedure (28) in that restriction sites placed at both ends of a double stranded DNA allow for the convenience of cloning each double stranded DNA fragment, individually, by (dC)-tailing and annealling into a (dG)-tailed vector, a method which in practice requires less of the double stranded DNA than three-part ligations.
Chemical Synthesis Eight fragments, 43, 43, 40, 4b, 46, 46, 54, and 4v bases in length (see Fig. 1), were chemically synthesized according to the method of Crea and horn (2), the only change being the use of mesitylene nitrotriazole as the condensing agent rather than 2,4,6 Triisopropyl benzenesulfonylchloride tetrazole.
The syntheses of the fragments were accomplished from the appropriate solid support (cellulose) by sequential addition of the appropriate fully-protected dimer- or trimer-blocks. The cycles i3 41491 were carried out under the same conditions as described in the synthesis of oligothymidilic acid (see Crea et al., supra). The final polymers were treated ;pith base (aqueous conc. NH3) and acid (80 percent HoAc), the polymer pelleted off, and the supernatant evaporated to dryness. The residue, dissolved in 4 percent aq.
NH3, was washed with ethyl ester (3X) and used for the isolation of the fully deprotectad fragment.
Purification was accomplished by electrophoresis using 20 percent polyacrylamide gels. The pure oligonucleotide was ethanol precipitated following gel elution.
225-285 pmoles of each chemically synthesized fragment was mixed with an equivalent amount of the complementary single-stranded DNA
~5 fragment (i.e. 1L+3L; 2L+4L; 1R+3R; 2R+4R) in the presence of deoxyri bonucl eosi de tri phosphates at a f i nal concantrati on of 2100 uv wi th the excepti on of dCTP. di,TP was added to a concentration of 5 uiv as a a32P-labeled isotope with a specific activity of 1000-2000 Ci/~unol) to allow easy monitoring of the repair-synthesis reaction 2p product. The reactions were carried out in a buffer containing a f i nal concentrati on of 50mM Tri s HC1 pH 7.5; 20 m~1 fAgCl 2; 20 m~~i DTT and 154 DNA Polymerase I (Klenow) in a reaction volume of 200 ul. Reactions were allowed to proceed at 4' for 12-18 hrs.
25 Upon completion, EDTA was added to a concentration of 25 mid.
Sample Suffer containing the mixes were phenol extracted, CHC13 extracted 2X, and products were etOH precipitated. Pellets were taken up in .3 M NaOAc and the DNA reprecipitated with etOH. Aft' r dissolving the pellets in H20, the 1L+3L and 2L+4L products were 30 then digested separately with PstI in 100 ul reaction mixes contai ni ng 1X PstI buffer ( 50mi~1 ( NH4) 2504, 20 miH Tri s HC1 pH
7.5, 10 mM MgCIZ), and 70 U PstI. After 4 hrs, EDTA ~,~as added to a concentration of 10 m"~I, and the material vas ethanol precipitated. Pellets were then taken up in .3 (~! NaOAc and 35 r2precipitated, then taken up in H20. The PstI-digested 1L+3L

product was digested with EcoRI at 37" in a 100 ul reaction mix 1X
EcoRI buffer ( 150 m~~t NaCI , 5 mil Tri s HC1 pH 7.5, 6 ru~i ~lgCl 2 ) and 70 U EcoRI. The Pstl digested 2L+4L product was digested at 37' with BamHI in a 100 ~1 reaction mix in 1X BamHI Buffer (150 mM NaCI, G m~~l Tri s HC1 pH 7.9, G mil ~IgCl 2) and 70 U BamHI. After 4 hrs, EDTA was added to both mixtures, and sample buffer was added. They were electrophoresed on a 6 percent polyacrylamide slab gel. Six percent slab gels were cast with a mixture containing 6 percent (w/v) acrylamide (20 to 1 ratio of acrylamide to Bis acrylamide) 1X
TBE, 1 percent APS and 0.1 percent TE~1ED. Reaction products were located on the gel by autoradiography and the oand corresponding to the ~5 by EcoRI-PstI digested 1L+3L product (Part 1) (see Fig. 3) and the band corresponding to the 50 by PstI-BamHI digested 2L+4L
product (Part 2) (see Fig. 3) were excised from the gel, the material electroeluted in 0.2X TBE, phenol extracted, CHC13 extracted, and ethanol preci pi tated. Parts 1 and 2 v~ere di ssol ve~.i i n H 20 .
Clonin3 Vector Prep.
Cloning vector eras prepared by digesting 20 ug pBR322 (15) with 50 U tcoRI and 50 U BamHI, in 1X RI Buffer at 37' For G hr. after addition of EDTA to a concentration of 10 mil, sample buffer was added, and the mixture was run on a 5 percent polyacrylamide gel.
The gel ivas developed by staining 10' in H20 containing 5 ug/ml Et. Bromide, rinsing 2X in H20 and placing upon a UY
transilluminator (302 n~~). the band corresponding to the ca. 3712 by EcoRi-BamHI digested pBR322 molecules was cut from the gel. 1'he~
DyA was electroeluted from the gel slice, phenol extracted, CHC13 extracted 2X, and ethanol precipitated. The pellet was dissolved in H20 and was ready for ligation.
Li q_ati on.
In a three-part ligation (see Fig. 4), in which the molar ratio of inserts to vector in the ligation reaction was approximately 10 to 1, parts 1 an:I 2 were ligated into the EcoRI-BamHI digested 322 vector in 1X T4 DNA lipase buffer (cont. 50 m~ Tris HC1 pH 7.8; 10 rciN i4gCl 2, 20 rvi~i DTT, I mM rATP ) and X800 U T4 D~dA 1 i gase ( NEB ) .
The reacti on was carri ed out at 14° for 12-lu hrs.
Transf ormati ons E. coli strain 294 was used as the transformation host, using the procedure of 'rvl. Dagert and S.D. Ehrlich (3). The transformed cells ~:rere plated on LB-agar plates containing ampicillin (20 ug/ml;
LB-Amp-plates) and transformants were screened and grown in LB
medium containing ampicillin at 20 ~g/ml ampicillin. Transformants were screened using a modification of the rapid miniscreen method of Birnboim and Doly (4). htiniprep DiJA prepared as such was digested wits EcoRI, and 3amHI and run on polyacrylamide slab gels. Several transf or~nants which illustrated a ca. 218 by EcoRI-BamHI insert were gro~vn i n 1 arge scal a and pl asmi ds from each were i sol ated and sequenced according to the procedure of ~taxam and Gilbert (5) to .confirm the correct chemical synthesis and construction. The pBR322 vector containing the complete correct left half sequence of IGF-1 vas called IGF-1 LH 322 (see Fig. 5).
Cloning of Fragments of the Right Half of IGF-1.
Using the identical conditions of DNA Polymerase I-mediated repair-synthesis, the two pairs of fragments comprising the right half of the synthetic IGF-1 were converted into double-stranded DNAs. After the DNA Polymerase I reactions, and without enzymatic digestion, the 1R+3R (Part III) and 2R+4R (Part IV) reactions were run on a 5 percent polyacrylamide slab gels. The 83 by (Part III) and 91 by (Part IV) bands were located by autoradiography and cut from the gel. After electroelution the ethanol precipitated double-stranded DNAs were dC-tailed (see Fig. 6) using the procedures of Villa-Komaroff et al. (b) and Rowenkamp and Firtel i~ 41491._ (7). Reactions were carried out in 50 ul vols. of 1X tailing mix (cont. .2~9 Pot. Cacodyl ate, 25 mil Tri s HC1 pH 5.9, 2 mil DTT, .5 m;~l CoCl2) and 22 um dCTP. After prewarning at 37° for 10', the 150 second reaction ;vas begun by the addition of 10-20 units of terminal nucleotidyl transferase and terminated by addition of EDTA followed by phenol extraction, CHC13 extraction 2X, and ethanol precipitation.
These oligo (dC) tailed Parts III and IV were then separately mixed with equimolar amounts of oligo (dG)-tailed PstI cut pBR322 vector i n 50 ul of 1X anneal l i ng buffer ( .1t~1 NaCI ; 10 m~1 Tri s HC1 pH
7.8, 1 mil EDTA) at a final DNA concentration of 1-2 ug/ml. After heating to 75°C, the mixes were gradually cooled to 4° over a period of lti hr and the mix transformed into competent E. coli 294 cells prepared according to the procedure of Dagert and Ehrlich (3).
Transformed cells were plated on L8-Tetracycl=,ne-Agar plates and grown in LB-Tetracycline medium at tetracycline ;.oncentrations of 5 ~g/ml. Tetracycline resistant transformants were picked and plated onto LB-Ampicillin-Agar plates to check for insertions at the PstI
.site. Several tetracycline resistant, Ampicillin-sensitive colonies for each Part 3 and r+ ~.vere mi ni screened and those exhi bi ti ng insertions at the PstI locus were grown in large scale and sequenced ~y the I~1axam and Gi 1 pert techni que ( 5) to conf i rm the correct DNA
sequences of Parts 3 and 4.
Construction of an Intact Synti~etic HuIGF-1 Coding Sequence Preparation: Parts 3 and 4.
Parts 3 and 4 :rere separately removed from their vectors by digestions of 20 ug of each vector with AvaII in 1X AvaII buffer (GO
mil NaCI , 6 mid Tri s-HC1 ( pH 8. 0 ) ; 10 m'1 ~IgCi 2; 6 mM
2-mercaptoethanol) and 30 U of AvaII. After o hr., at 37', EDTA was added to the 150 ul reactions to a concentration of 15 min and the material phenol extracted, CHC13 extracted 2X and ethanol 1s 41491 precipitated. The Part 3 pellet was then taken up in 1X BamHI
buffer and digested in a volume of 150 ul with 30 U BamHI at 37° for 4 h r. The pellet containing Part 4 was digested with 30 U SaII in 150 ul of 1X SalI buffer at 31° for 4 hr.
Both digests were then run on 6 percent polyacrylamide slab gels and stained. The 5i by band representing Part 3 and the G2 by band representing Part 4 were removed from the gels and the DNA
electroeluted, phenol extracted, CHC13 extracted 2X and ethanol precipitated. Pellets were then taken up in H20 and were ready for ligation.
Vector Preparation 20 Ng of the IGF-1 LH 322 vector was digested with 50 U of BamHI
and 50 U o.f SaII in a 200 ,~1 reaction contain~~~g 1X BamHI buffer at 37" for 5 hr. After addition of EDTA to a concentration of 15 mM, the digestion mix was run on a o percent polyacrylamide slab gel, ethidium bromide stained and the 3814 by band excised from the gel.
After electroelution, phenol extraction, chloroform extraction and ethanol precipitation, the DNA pellet was taken up in H20 and was ready for ligation with Parts 3 and 4 in a three-part ligation.
The 1 i gati on teas performed under condi ti ons descri bed above for a three-part ligation isee Fig. 7). Parts 3 and 4 were present in the ligation mix at a 10-fold molar excess of inserts to vector. The mix was transformed into competent E. coli 294 cells prepared according to the Dagert and Ehrlich procedure (3) and plated onto -LB-Ampicillin plates. Several transformants were miniscreened and two clones exhibiting a ca. 115 by BamHI-SaII fragment were grown in large scale and their plasmids prepared. Both strands of the intact synthetic gene were sequenced by the Maxam-Gilbert technique (5) to confir;n the correct sequence. The pBR322 plasmid containing the complete correct sequence coding for Human IGF-1 was called paR322 HuIGF-1.

1 ~ 4 'I 4 ~ 1 Human IGF-1 Expression IGF-1 Fusion Expression in Bacteria Initial attempts :sere to obtain expression of IGF-1 as a fusion protein. To accomplish this, both t~~e pNCV (9) and the pNCVsLE (10) expression vectors were used. (The pNCVsLE expression vector is a derivative of the pNCV vector and :gas prepared as follows: pNCY ~~as treated with BgII, which cleaves at the 13 codon of the LE fusion.
The site was converted to an ECoRl cleavage site using synthetic DNA, to give the expression vector ptdCVsLE. The synthetic DNA
introduced into the plasmid has the sequence:

7. Rowenkamp and sirtel, Dictyostelium Dev. Biol. 79, 409 (1980).

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Claims

1. A process for producing mature human insulin like growth factor II having the amino acid sequence Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Ser Arg Pro Ala Ser Arg Val Ser Arg Arg Ser Arg Gly Ile Val Glu Glu Cys Cys Phe Arg Ser Cys Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala Thr Pro Ala Lys Ser Glu, comprising (a) transforming a yeast host cell with a replicable expression vector comprising a DNA sequence encoding said growth factor in proper reading frame with secretory processing and signal sequences derived from yeast .alpha.-factor DNA, and operably associated with expression control elements functional in said yeast host cell, (b) culturing the transformed host cell of (a) under conditions permitting expression of said growth factor and secretion into the culture medium, and (c) recovering said growth factor from the culture medium.

2. A yeast host cell transformed with a replicable expression vector comprising a DNA sequence encoding mature human insulin like growth factor II having the amino acid sequence Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Try Phe Ser Arg Pro Ala Ser Arg Val Ser Arg Arg Ser Arg Gly Ile Val Glu Glu Cys Cys Phe Arg Ser Cys Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala Thr Pro Ala Lys Ser Glu, in proper reading frame with secretory processing and signal sequences derived from yeast .alpha.-Factor DNA, and operably associated with expression control elements functional in said yeast host cell.

3. A replicable expression vector comprising a DNA
sequence encoding mature human insulin like growth factor II having the amino acid sequence Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Ser Arg Pro Ala Ser Arg Val Ser Arg Arg Ser Arg Gly Ile Val Glu Glu Cys Cys Phe Arg Ser Cys Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala Thr Pro Ala Lys Ser Glu, in proper reading frame with secretory processing and signal sequences derived from yeast .alpha.-Factor DNA, and operably associated with expression control elements functional in said yeast host cell.