TABLE 1


Original	Exemplary	Preferred
Residue	Substitutions	Substitutions

Ala (A)	val; leu; ile	val
Arg (R)	lys; gln; asn	lys
Asn (N)	gln; his; lys; arg	gln
Asp (D)	glu	glu
Cys (C)	ser	ser
Gln (Q)	asn	asn
Glu (E)	asp	asp
Gly (G)	pro	pro
His (H)	asn; gln; lys; arg	arg
Ile (I)	leu; val; met; ala; phe;
	norleucine	leu
Leu (L)	norleucine; ile; val;
	met; ala; phe	ile
Lys (K)	arg; gln; asn	arg
Met (M)	leu; phe; ile	leu
Phe (F)	leu; val; ile; ala	leu
Pro (P)	gly	gly
Ser (S)	thr	thr
Thr (T)	ser	ser
Trp (W)	tyr	tyr
Tyr (Y)	trp; phe; thr; ser	phe
Val (V)	ile; leu; met; phe;
	ala; norleucine	leu

Substantial modifications in function or immunological identity of the TSF receptor are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties:[0089]

(1) hydrophobic: norleucine, met, ala, val, leu, ile;[0090]

(2) neutral hydrophilic: cys, ser, thr;[0091]

(3) acidic: asp, glu;[0092]

(4) basic: asn, gln, his, lys, arg;[0093]

(5) residues that influence chain orientation: gly, pro; and[0094]

(6) aromatic: trp, tyr, phe.[0095]

Non-conservative substitutions will entail exchanging a member of one of these classes for another. Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.[0096]

In one embodiment of the invention, it is desirable to inactivate one or more protease cleavage sites that are present in the molecule. These sites are identified by inspection of the encoded amino acid sequence, in the case of trypsin, e.g., for an arginyl or lysinyl residue. When protease cleavage sites are identified, they are rendered inactive to proteolytic cleavage by substituting the targeted residue with another residue, preferably a basic residue such as glutamine or a hydrophobic residue such as serine; by deleting the residue; or by inserting a prolyl residue immediately after the residue.[0097]

In another embodiment, any methionyl residues other than the starting methionyl residue of the signal sequence, or any residue located within about three residues N- or C-terminal to each such methionyl residue, is substituted by another residue (preferably in accord with Table 1) or deleted. Alternatively, about 1-3 residues are inserted adjacent to such sites.[0098]

Any cysteine residues not involved in maintaining the proper conformation of the TSF receptor also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.[0099]

Nucleic acid molecules encoding amino acid sequence variants of TSF receptor are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) nutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of TSF receptor polypeptide.[0100]

Oligonucleotide-mediated mutagenesis is a preferred method for preparing substitution, deletion, and insertion variants of TSF receptor DNA. This technique is well known in the art as described by Adelman et al.,[0101]DNA,2: 183 (1983). Briefly, TSF receptor DNA is altered by hybridizing an oligonucleotide encoding the desired mutation to a DNA template, where the template is the single-stranded form of a plasmid or bacteriophage containing the unaltered or native DNA sequence of TSF receptor. After hybridization, a DNA polymerase is used to synthesize an entire second complementary strand of the template that will thus incorporate the oligonucleotide primer, and will code for the selected alteration in the TSF receptor DNA.

Generally, oligonucleotides of at least 25 nucleotides in length are used. An optimal oligonucleotide will have 12 to 15 nucleotides that are completely complementary to the template on either side of the nucleotide(s) coding for the mutation. This ensures that the oligonucleotide will hybridize properly to the single-stranded DNA template molecule. The oligonucleotides are readily synthesized using techniques known in the art such as that described by Crea et al.,[0102]Proc. Natl. Acad. Sci. USA,75: 5765 (1978).

The DNA template can be generated by those vectors that are either derived from bacteriophage M13 vectors (the commercially available M13 mp18 and M13 mp19 vectors are suitable), or those vectors that contain a single-stranded phage origin of replication as described by Viera et al.[0103]Meth. Enzymol.,153: 3 (1987). Thus, the DNA that is to be mutated may be inserted into one of these vectors to generate single-stranded template. Production of the single-stranded template is described in Sections 4.21-4.41 of Sambrook et al.,Molecular Cloning: A Laboratory Manual(Cold Spring Harbor Laboratory Press, NY 1989).

Alternatively, single-stranded DNA template may be generated by denaturing double-stranded plasmid (or other) DNA using standard techniques.[0104]

For alteration of the native DNA sequence (to generate amino acid sequence variants, for example), the oligonucleotide is hybridized to the single-stranded template under suitable hybridization conditions. A DNA polymerizing enzyme, usually the Klenow fragment of DNA polymerase I, is then added to synthesize the complementary strand of the template using the oligonucleotide as a primer for synthesis. A heteroduplex molecule is thus formed such that one strand of DNA encodes the mutated form of the TSF receptor, and the other strand (the original template) encodes the native, unaltered sequence of the TSF receptor. This heteroduplex molecule is then transformed into a suitable host cell, usually a prokaryote such as[0105]E. coliJM101. After the cells are grown, they are plated onto agarose plates and screened using the oligonucleotide primer radiolabeled with 32-phosphate to identify the bacterial colonies that contain the mutated DNA. The mutated region is then removed and placed in an appropriate vector for protein production, generally an expression vector of the type typically employed for transformation of an appropriate host.

The method described immediately above may be modified such that a homoduplex molecule is created wherein both strands of the plasmid contain the mutation(s). The modifications are as follows: The single-stranded oligonucleotide is annealed to the single-stranded template as described above. A mixture of three deoxyribonucleotides, deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), and deoxyribothymidine (dTTP), is combined with a modified thio-deoxyribocytosine called dCTP-(aS) (which can be obtained from the Amersham Corporation). This mixture is added to the template-oligonucleotide complex. Upon addition of DNA polymerase to this mixture, a strand of DNA identical to the template except for the mutated bases is generated. In addition, this new strand of DNA will contain dCTP-(aS) instead of dCTP, which serves to protect it from restriction endonuclease digestion.[0106]

After the template strand of the double-stranded heteroduplex is nicked with an appropriate restriction enzyme, the template strand can be digested with ExoIII nuclease or another appropriate nuclease past the region that contains the site(s) to be nutagenized. The reaction is then stopped to leave a molecule that is only partially single-stranded. A complete double-stranded DNA homoduplex is then formed using DNA polymerase in the presence of all four deoxyribonucleotide triphosphates, ATP, and DNA ligase. This homoduplex molecule can then be transformed into a suitable host cell such as[0107]E. coliJM101, as described above.

DNA encoding TSF receptor mutants with more than one amino acid to be substituted may be generated in one of several ways. If the amino acids are located close together in the polypeptide chain, they may be mutated simultaneously using one oligonucleotide that codes for all of the desired amino acid substitutions. If, however, the amino acids are located some distance from each other (separated by more than about ten amino acids), it is more difficult to generate a single oligonucleotide that encodes all of the desired changes. Instead, one of two alternative methods may be employed.[0108]

In the first method, a separate oligonucleotide is generated for each amino acid to be substituted. The oligonucleotides are then annealed to the single-stranded template DNA simultaneously, and the second strand of DNA that is synthesized from the template will encode all of the desired amino acid substitutions.[0109]

The alternative method involves two or more rounds of mutagenesis to produce the desired mutant. The first round is as described for the single mutants: wild-type DNA is used for the template, an oligonucleotide encoding the first desired amino acid substitution(s) is annealed to this template, and the heteroduplex DNA molecule is then generated. The second round of mutagenesis utilizes the mutated DNA produced in the first round of mutagenesis as the template. Thus, this template already contains one or more mutations. The oligonucleotide encoding the additional desired amino acid substitution(s) is then annealed to this template, and the resulting strand of DNA now encodes mutations from both the first and second rounds of mutagenesis. This resultant DNA can be used as a template in a third round of mutagenesis, and so on.[0110]

PCR mutagenesis is also suitable for making amino acid variants of TSF receptor polypeptide. While the following discussion refers to DNA, it is understood that the technique also finds application with RNA. The PCR technique generally refers to the following procedure (see Erlich, supra, the chapter by R. Higuchi, p. 61-70): When small amounts of template DNA are used as starting material in a PCR, primers that differ slightly in sequence from the corresponding region in a template DNA can be used to generate relatively large quantities of a specific DNA fragment that differs from the template sequence only at the positions where the primers differ from the template. For introduction of a mutation into a plasmid DNA, one of the primers is designed to overlap the position of the mutation and to contain the mutation; the sequence of the other primer must be identical to a stretch of sequence of the opposite strand of the plasmid, but this sequence can be located anywhere along the plasmid DNA. It is preferred, however, that the sequence of the second primer is located within 200 nucleotides from that of the first, such that in the end the entire amplified region of DNA bounded by the primers can be easily sequenced. PCR amplification using a primer pair like the one just described results in a population of DNA fragments that differ at the position of the mutation specified by the primer, and possibly at other positions, as template copying is somewhat error-prone.[0111]

If the ratio of template to product material is extremely low, the vast majority of product DNA fragments incorporate the desired mutation(s). This product material is used to replace the corresponding region in the plasmid that served as PCR template using standard DNA technology. Mutations at separate positions can be introduced simultaneously by either using a mutant second primer, or performing a second PCR with different mutant primers and ligating the two resulting PCR fragments simultaneously to the vector fragment in a three (or more)-part ligation.[0112]

In a specific example of PCR mutagenesis, template plasmid DNA (1 μg) is linearized by digestion with a restriction endonuclease that has a unique recognition site in the plasmid DNA outside of the region to be amplified. Of this material, 100 ng is added to a PCR mixture containing PCR buffer, which contains the four deoxynucleotide triphosphates and is included in the GeneAmp® kits (obtained from Perkin-Elmer Cetus, Norwalk, Conn. and Emeryville, Calif.), and 25 pmole of each oligonucleotide primer, to a final volume of 50 μl. The reaction mixture is overlayed with 35 μl mineral oil. The reaction mixture is denatured for five minutes at 100° C., placed briefly on ice, and then 1 μl[0113]Thermus aquaticus(Taq) DNA polymerase (5 units/μl, purchased from Perkin-Elmer Cetus) is added below the mineral oil layer. The reaction mixture is then inserted into a DNA Thermal Cycler (purchased from Perkin-Elmer Cetus) programmed as follows:

2 min. 55° C.[0114]

30 sec. 72° C., then 19 cycles of the following:[0115]

30 sec. 94° C.[0116]

30 sec. 55° C., and[0117]

30 sec. 72° C.[0118]

At the end of the program, the reaction vial is removed from the thermal cycler and the aqueous phase transferred to a new vial, extracted with phenol/chloroform (50:50 vol), and ethanol precipitated, and the DNA is recovered by standard procedures. This material is subsequently subjected to the appropriate treatments for insertion into a vector.[0119]

Another method for preparing variants, cassette mutagenesis, is based on the technique described by Wells et al.,[0120]Gene,34: 315 (1985). The starting material is the plasmid (or other vector) comprising the TSF receptor DNA to be mutated. The codon(s) in the TSF receptor DNA to be mutated are identified. There must be a unique restriction endonuclease site on each side of the identified mutation site(s). If no such restriction sites exist, they may be generated using the above-described oligonucleotide-mediated mutagenesis method to introduce them at appropriate locations in the TSF receptor DNA. After the restriction sites have been introduced into the plasmid, the plasmid is cut at these sites to linearize it. A double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures. The two strands are synthesized separately and then hybridized together using standard techniques. This double-stranded oligonucleotide is referred to as the cassette. This cassette is designed to have 3′ and 5′ ends that are compatible with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid. This plasmid now contains the mutated TSF receptor DNA sequence.

C. Insertion of Nucleic Acid into a Replicable Vector[0121]

The nucleic acid (e.g., cDNA or genomic DNA) encoding native or variant TSF receptor polypeptide is inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. Many vectors are available, and selection of the appropriate vector will depend on 1) whether it is to be used for DNA amplification or for DNA expression, 2) the size of the nucleic acid to be inserted into the vector, and 3) the host cell to be transformed with the vector. Each vector contains various components depending on its function (amplification of DNA or expression of DNA) and the host cell with which it is compatible. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.[0122]

(i) Signal Sequence Component[0123]

The TSF receptor of this invention may be expressed not only directly, but also as a fusion with a heterologous polypeptide, preferably a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of the vector, or it may be a part of the TSF receptor DNA that is inserted into the vector. The heterologous signal sequence selected should be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the native TSF receptor signal sequence, the signal sequence is substituted by a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion the native signal sequence may be substituted by, e.g., the yeast invertase, alpha factor, or acid phosphatase leaders, the[0124]C. albicansglucoamylase leader (EP 362,179 published Apr. 4, 1990), or the signal described in WO 90/13646 published Nov. 15, 1990. In mammalian cell expression the native signal sequence (i.e., the TSF receptor presequence that normally directs secretion of TSF receptor from its naitve mammalian cells in vivo) is satisfactory, although other mammalian signal sequences may be suitable, such as signal sequences from other TSF receptor polypeptides or from the same TSF receptor from a different animal species, signal sequences from a TSF member, including inhibin or activin, and signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders, for example, the herpes simplex gD signal.

(ii) Origin of Replication Component[0125]

Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Generally, in cloning vectors this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not needed for mammalian expression vectors (the SV40 origin may typically be used only because it contains the early promoter).[0126]

Most expression vectors are “shuttle” vectors, i.e., they are capable of replication in at least one class of organisms but can be transfected into another organism for expression. For example, a vector is cloned in[0127]E. coliand then the same vector is transfected into yeast or mammalian cells for expression even though it is not capable of replicating independently of the host cell chromosome.

DNA may also be amplified by insertion into the host genome. This is readily accomplished using Bacillus species as hosts, for example, by including in the vector a DNA sequence that is complementary to a sequence found in Bacillus genomic DNA. Transfection of Bacillus with this vector results in homologous recombination with the genome and insertion of TSF receptor DNA. However, the recovery of genomic DNA encoding TSF receptor is more complex than that of an exogenously replicated vector because restriction enzyme digestion is required to excise the TSF receptor DNA.[0128]

(iii) Selection Gene Component[0129]

Expression and cloning vectors should contain a selection gene, also termed a selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.[0130]

One example of a selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene express a protein conferring drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs neomycin (Southern et al.,[0131]J. Molec. Appl. Genet.,1: 327 [1982]), mycophenolic acid (Mulligan et al.,Science,209: 1422 [1980]) or hygromycin (Sugden et al.,Mol. Cell. Biol.,5: 410-413 [1985]). The three examples given above employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid), or hygromycin, respectively.

Another example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the TSF receptor nucleic acid, such as dihydrofolate reductase (DHFR) or thymidine kinase. The mammalian cell transformants are placed under selection pressure that only the transformants are uniquely adapted to survive by virtue of having taken up the marker. Selection pressure is imposed by culturing the transformants under conditions in which the concentration of selection agent in the medium is successively changed, thereby leading to amplification of both the selection gene and the DNA that encodes TSF receptor polypeptide. Amplification is the process by which genes in greater demand for the production of a protein critical for growth are reiterated in tandem within the chromosomes of successive generations of recombinant cells. Increased quantities of TSF receptor are synthesized from the amplified DNA.[0132]

For example, cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR. An appropriate host cell when wild-type DHFR is employed is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity, prepared and propagated as described by Urlaub and Chasin,[0133]Proc. Natl. Acad. Sci. USA,77: 4216 [1980]. The transformed cells are then exposed to increased levels of Mtx. This leads to the synthesis of multiple copies of the DHFR gene, and, concomitantly, multiple copies of other DNA comprising the expression vectors, such as the DNA encoding TSF receptor. This amplification technique can be used with any otherwise suitable host, e.g., ATCC No. CCL61 CHO-K1, notwithstanding the presence of endogenous DHFR if, for example, a mutant DHFR gene that is highly resistant to Mtx is employed (EP 117,060). Alternatively, host cells [particularly wild-type hosts that contain endogenous DHFR] transformed or co-transformed with DNA sequences encoding TSF receptor, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3′ phosphotransferase (APH) can be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present in the yeast plasmid YRp7 (Stinchcomb et al.,[0134]Nature,282: 39 [1979]; Kingsman et al.,Gene,7: 141 [1979]; or Tschemper et al.,Gene,10: 157 [1980]). The trp1 gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 (Jones,Genetics,85: 12 [1977]). The presence of the trp1 lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) are complemented by known plasmids bearing the Leu2 gene.

(iv) Promoter Component[0135]

Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the TSF receptor nucleic acid. Promoters are untranslated sequences located upstream (5′) to the start codon of a structural gene (generally within about 100 to 1000 bp) that control the transcription and translation of particular nucleic acid sequence, such as the TSF receptor nucleic acid sequence, to which they are operably linked. Such promoters typically fall into two classes, inducible and constitutive. Inducible promoters are promoters that initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, e.g., the presence or absence of a nutrient or a change in temperature. At this time a large number of promoters recognized by a variety of potential host cells are well known. These promoters are operably linked to TSF-receptor-encoding DNA by removing the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector. Both the native TSF receptor promoter sequence and many heterologous promoters may be used to direct amplification and/or expression of the TSF receptor DNA. However, heterologous promoters are preferred, as they generally permit greater transcription and higher yields of expressed TSF receptor as compared to the native TSF receptor promoter.[0136]

Promoters suitable for use with prokaryotic hosts include the β-lactamase and lactose promoter systems (Chang et al.,[0137]Nature,275: 615 [1978]; and Goeddel et al.,Nature,281: 544 [1979]), alkaline phosphatase, a tryptophan (trp) promoter system (Goeddel,Nucleic Acids Res.,8: 4057 [1980] and EP 36,776) and hybrid promoters such as the tac promoter (deBoer et al.,Proc. Natl. Acad. Sci. USA,80: 21-25 [1983]). However, other known bacterial promoters are suitable. Their nucleotide sequences have been published, thereby enabling a skilled worker operably to ligate them to DNA encoding TSF receptor (Siebenlist et al.,Cell,20: 269 [1980]) using linkers or adaptors to supply any required restriction sites. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding TSF receptor polypeptide.

Promoter sequences are known for eukaryotes. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CXCAAT region where X may be any nucleotide. At the 3′ end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of the poly A tail to the 3′ end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors.[0138]

Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase (Hitzeman et al.,[0139]J. Biol. Chem.,255: 2073 [1980]) or other glycolytic enzymes (Hess et al.,J. Adv. Enzyme Reg.,7: 149 [1968]; and Holland,Biochemistry,17: 4900 [1978]), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in Hitzeman et al., EP 73,657A. Yeast enhancers also are advantageously used with yeast promoters.[0140]

TSF receptor transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published Jul. 5, 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, from heat-shock promoters, and from the promoter normally associated with the TSF receptor sequence, provided such promoters are compatible with the host cell systems.[0141]

The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. Fiers et al.,[0142]Nature,273:113 (1978); Mulligan and Berg,Science,209: 1422-1427 (1980); Pavlakis et al.,Proc. Natl. Acad. Sci. USA,78: 7398-7402 (1981). The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment. Greenaway et al.,Gene,18: 355-360 (1982). A system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,446. A modification of this system is described in U.S. Pat. No. 4,601,978. See also Gray et al.,Nature,295: 503-508 (1982) on expressing cDNA encoding immune interferon in monkey cells; Reyes et al.,Nature,297: 598-601 (1982) on expression of human β-interferon cDNA in mouse cells under the control of a thymidine kinase promoter from herpes simplex virus; Canaani and Berg,Proc. Natl. Acad. Sci. USA,79: 5166-5170 (1982) on expression of the human interferon β1 gene in cultured mouse and rabbit cells; and Gorman et al.,Proc. Natl. Acad. Sci. USA,79: 6777-6781 (1982) on expression of bacterial CAT sequences in CV-1 monkey kidney cells, chicken embryo fibroblasts, Chinese hamster ovary cells, HeLa cells, and mouse NIH-3T3 cells using the Rous sarcoma virus long terminal repeat as a promoter.

(v) Enhancer Element Component[0143]

Transcription of a DNA encoding the TSF receptor of this invention by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Enhancers are relatively orientation and position independent, having been found 5′ (Laimins et al.,[0144]Proc. Natl. Acad. Sci. USA,78: 993 [1981]) and 3′ (Lusky et al.,Mol. Cell Bio.,3: 1108 [1983]) to the transcription unit, within an intron (Banerji et al.,Cell,33: 729 [1983]), as well as within the coding sequence itself (Osborne et al.,Mol. Cell Bio.,4: 1293 [1984]). Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv,Nature,297: 17-18 (1982) on enhancing elements for activation of eukaryotic promoters. The enhancer may be spliced into the vector at a position 5′ or 3′ to the TSF-receptor-encoding sequence, but is preferably located at a site 5′ from the promoter.

(vi) Transcription Termination Component[0145]

Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5′ and, occasionally 3′ untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding TSF receptor.[0146]

(vii) Construction and Analysis of Vectors[0147]

Construction of suitable vectors containing one or more of the above listed components employs standard ligation techniques. Isolated plasmids or DNA fragments are cleaved, tailored, and religated in the form desired to generate the plasmids required.[0148]

For analysis to confirm correct sequences in plasmids constructed, the ligation mixtures are used to transform[0149]E. coliK12 strain 294 (ATCC 31,446) and successful transformants selected by ampicillin or tetracycline resistance where appropriate. Plasmids from the transformants are prepared, analyzed by restriction endonuclease digestion, and/or sequenced by the method of Messing et al.,Nucleic Acids Res.,9: 309 (1981) or by the method of Maxam et al.,Methods in Enzymology,65: 499 (1980).

(viii) Transient Expression Vectors[0150]

Particularly useful in the practice of this invention are expression vectors that provide for the transient expression in mammalian cells of DNA encoding the TSF receptor polypeptide. In general, transient expression involves the use of an expression vector that is able to replicate efficiently in a host cell, such that the host cell accumulates many copies of the expression vector and, in turn, synthesizes high levels of a desired polypeptide encoded by the expression vector. Sambrook et al., supra, pp. 16.17-16.22. Transient expression systems, comprising a suitable expression vector and a host cell, allow for the convenient positive identification of polypeptides encoded by cloned DNAs, as well as for the rapid screening of such polypeptides for desired biological or physiological properties. Thus, transient expression systems are particularly useful in the invention for purposes of identifying analogs and variants of TSF receptor polypeptide that have TSF receptor polypeptide biological activity.[0151]

(ix) Suitable Exemplary Vertebrate Cell Vectors[0152]

Other methods, vectors, and host cells suitable for adaptation to the synthesis of TSF receptor in recombinant vertebrate cell culture are described in Gething et al.,[0153]Nature,293: 620-625 [1981]; Mantei et al.,Nature,281: 40-46 [1979]; Levinson et al.; EP 117,060; and EP 117,058. A particularly useful plasmid for mammalian cell culture expression of TSF receptor is pRK5 (EP pub. no. 307,247) or pSVI6B (U.S. Ser. No. 07/441,574 filed Nov. 22, 1989, the disclosure of which is incorporated herein by reference).

D. Selection and Transformation of Host Cells[0154]

Suitable host cells for cloning or expressing the vectors herein are the prokaryote, yeast, or higher eukaryotic cells described above. Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-positive organisms, for example,[0155]E. coli, Bacilli such asB. subtilis, Pseudomonas species such asP. aeruginosa, Salmonella typhimurium, orSerratia marcescans. One preferredE. colicloning host isE. coli294 (ATCC 31,446), although other strains such asE. coliB,E. coliX1776 (ATCC 31,537), andE. coliW3110 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting. Preferably the host cell should secrete minimal amounts of proteolytic enzymes. Alternatively, in vitro methods of cloning, e.g., PCR or other nucleic acid polymerase reactions, are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable hosts for TSF-receptor-encoding vectors.[0156]Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and useful herein, such asSchizosaccharomyces pombe[Beach and Nurse,Nature,290: 140 (1981); EP 139,383 published May 2, 1985], Kluyveromyces hosts (U.S. Pat. No. 4,943,529) such as, e.g.,K. lactis[Louvencourt et al.,J. Bacteriol.,737 (1983)],K. fragilis, K. bulgaricus, K. thermotolerans, andK. marxianus, yarrowia [EP 402,226],Pichia pastoris[EP 183,070; Sreekrishna et al.,J. Basic Microbiol.,28: 265-278 (1988)], Candida,Trichoderma reesia[EP 244,234],Neurospora crassa[Case et al.,Proc. Natl. Acad. Sci. USA,76: 5259-5263 (1979)], and filamentous fungi such as, e.g, Neurospora, Penicillium, Tolypocladium [WO 91/00357 published Jan. 10, 1991], and Aspergillus hosts such asA. nidulans[Ballance et al.,Biochem. Biophys. Res. Commun.,112: 284-289 (1983); Tilburn et al.,Gene,26: 205-221 (1983); Yelton et al.,Proc. Natl. Acad. Sci. USA,81: 1470-1474 (1984)] andA. niger[Kelly and Hynes,EMBO J.,4: 475-479 (1985)].

Suitable host cells for the expression of glycosylated TSF receptor are derived from multicellular organisms. Such host cells are capable of complex processing and glycosylation activities. In principle, any higher eukaryotic cell culture is workable, whether from vertebrate or invertebrate culture. Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as[0157]Spodoptera frugiperda(caterpillar),Aedes aegypti(mosquito),Aedes albopictus(mosquito),Drosophila melanogaster(fruitfly), andBombyx morihave been identified. See, e.g., Luckow et al.,Bio/Technology,6: 47-55 (1988); Miller et al., inGenetic Engineering, Setlow, J. K. et al., eds., Vol. 8 (Plenum Publishing, 1986), pp. 277-279; and Maeda et al.,Nature,315: 592-594 (1985). A variety of viral strains for transfection are publicly available, e.g., the L-1 variant ofAutographa californicaNPV and the Bm-5 strain ofBombyx moriNPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection ofSpodoptera frugiperdacells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can be utilized as hosts. Typically, plant cells are transfected by incubation with certain strains of the bacterium[0158]Agrobacterium tumefaciens, which has been previously manipulated to contain the TSF receptor DNA. During incubation of the plant cell culture withA. tumefaciens, the DNA encoding the TSF receptor is transferred to the plant cell host such that it is transfected, and will, under appropriate conditions, express the TSF receptor DNA. In addition, regulatory and signal sequences compatible with plant cells are available, such as the nopaline synthase promoter and polyadenylation signal sequences. Depicker et al.,J. Mol. Appl. Gen.,1: 561 (1982). In addition, DNA segments isolated from the upstream region of the T-DNA 780 gene are capable of activating or increasing transcription levels of plant-expressible genes in recombinant DNA-containing plant tissue. EP 321,196 published Jun. 21, 1989.

However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure in recent years [[0159]Tissue Culture, Academic Press, Kruse and Patterson, editors (1973)]. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al.,J. Gen Virol.,36: 59 [1977]); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin,Proc. Natl. Acad. Sci. USA,77: 4216 [1980]); mouse sertoli cells (TM4, Mather,Biol. Reprod.,23: 243-251 [1980]); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al.,Annals N.Y. Acad. Sci.,383: 44-68 [1982]); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

Host cells are transfected and preferably transformed with the above-described expression or cloning vectors of this invention and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.[0160]

Transfection refers to the taking up of an expression vector by a host cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, CaPO[0161]₄and electroporation. Successful transfection is generally recognized when any indication of the operation of this vector occurs within the host cell.

Transformation means introducing DNA into an organism so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integrant. Depending on the host cell used, transformation is done using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described in section 1.82 of Sambrook et al., supra, is generally used for prokaryotes or other cells that contain substantial cell-wall barriers. Infection with[0162]Agrobacterium tumefaciensis used for transformation of certain plant cells, as described by Shaw et al.,Gene,23: 315 (1983) and WO 89/05859 published Jun. 29, 1989. In addition, plants may be transfected using ultrasound treatment as described in WO 91/00358 published Jan. 10, 1991. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb,Virology,52: 456-457 (1978) is preferred. General aspects of mammalian cell host system transformations have been described by Axel in U.S. Pat. No. 4,399,216 issued Aug. 16, 1983. Transformations into yeast are typically carried out according to the method of Van Solingen et al.,J. Bact.,130: 946 (1977) and Hsiao et al.,Proc. Natl. Acad. Sci. (USA),76: 3829 (1979). However, other methods for introducing DNA into cells such as by nuclear injection, electroporation, or protoplast fusion may also be used.

E. Culturing the Host Cells[0163]

Prokaryotic cells used to produce the TSF receptor polypeptide of this invention are cultured in suitable media as described generally in Sambrook et al., supra.[0164]

The mammalian host cells used to produce the TSF receptor of this invention may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ([MEM], Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ([DMEM], Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham and Wallace,[0165]Meth. Enz.,58: 44 (1979), Barnes and Sato,Anal. Biochem.,102: 255 (1980), U.S. Pat. No. 4,767,704; 4,657,866; 4,927,762; or 4,560,655; WO 90/03430; WO 87/00195; U.S. Pat. No. Re. 30,985; or copending U.S. Ser. No. 07/592,107 or 07/592,141, both filed in Oct. 3, 1990, the disclosures of all of which are incorporated herein by reference, may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics (such as Gentamycin™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

The host cells referred to in this disclosure encompass cells in in vitro culture as well as cells that are within a host animal.[0166]

F. Detecting Gene Amplification/Expression[0167]

Gene amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, northern blotting to quantitate the transcription of mRNA (Thomas,[0168]Proc. Natl. Acad. Sci. USA,77: 5201-5205 [1980]), dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Various labels may be employed, most commonly radioisotopes, particularly³²P. However, other techniques may also be employed, such as using biotin-modified nucleotides for introduction into a polynucleotide. The biotin then serves as the site for binding to avidin or antibodies, which may be labeled with a wide variety of labels, such as radionuclides, fluorescers, enzymes, or the like. Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.

Gene expression, alternatively, may be measured by immunological methods, such as immunohistochemical staining of tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product. With immunohistochemical staining techniques, a cell sample is prepared, typically by dehydration and fixation, followed by reaction with labeled antibodies specific for the gene product coupled, where the labels are usually visually detectable, such as enzymatic labels, fluorescent labels, luminescent labels, and the like. A particularly sensitive staining technique suitable for use in the present invention is described by Hsu et al.,[0169]Am. J. Clin. Path.,75: 734-738 (1980).

Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the antibodies may be prepared against a native TSF receptor polypeptide or against a synthetic peptide based on the DNA sequences provided herein as described further in Section 3 below.[0170]

G. Purification of TSF Receptor Polypeptide[0171]

TSF receptor preferably is recovered from the culture medium as a secreted polypeptide, although it also may be recovered from host cell lysates when directly expressed without a secretory signal.[0172]

When TSF receptor is expressed in a recombinant cell other than one of human origin, the TSF receptor is completely free of proteins or polypeptides of human origin. However, it is necessary to purify TSF receptor from recombinant cell proteins or polypeptides to obtain preparations that are substantially homogeneous as to TSF receptor. As a first step, the culture medium or lysate is centrifuged to remove particulate cell debris. The membrane and soluble protein fractions are then separated. The TSF receptor may then be purified from the soluble protein fraction and from the membrane fraction of the culture lysate, depending on whether the TSF receptor is membrane bound. TSF receptor thereafter is purified from contaminant soluble proteins and polypeptides, with the following procedures being exemplary of suitable purification procedures: by fractionation on immunoaffinity or ion-exchange columns; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; ligand affinity chromatography, using, e.g., a TSF member as ligand, and protein A Sepharose columns to remove contaminants such as IgG.[0173]

TSF receptor variants in which residues have been deleted, inserted, or substituted are recovered in the same fashion as native TSF receptor, taking account of any substantial changes in properties occasioned by the variation. For example, preparation of a TSF receptor fusion with another protein or polypeptide, e.g., a bacterial or viral antigen, facilitates purification; an immunoaffinity column containing antibody to the antigen can be used to adsorb the fusion polypeptide. Immunoaffinity columns such as a rabbit polyclonal anti-TSF-receptor column can be employed to absorb the TSF receptor variant by binding it to at least one remaining immune epitope. Alternatively, the receptor molecule may be purified by affinity chromatography using a purified TSF member such as inhibin or activin as a ligand coupled to a (preferably) immobilized resin such as Affi-Gel 10 (Bio-Rad, Richmond, Calif.) or the like, by means well known in the art. A protease inhibitor such as phenyl methyl sulfonyl fluoride (PMSF) also may be useful to inhibit proteolytic degradation during purification, and antibiotics may be included to prevent the growth of adventitious contaminants. One skilled in the art will appreciate that purification methods suitable for native TSF receptor may require modification to account for changes in the character of TSF receptor or its variants upon expression in recombinant cell culture.[0174]

H. Covalent Modifications of TSF Receptor Polypeptide[0175]

Covalent modifications of TSF receptor polypeptides are included within the scope of this invention. Both native TSF receptor and amino acid sequence variants of the TSF receptor may be covalently modified. One type of covalent modification included within the scope of this invention is a TSF receptor fragment. Variant TSF receptor fragments having up to about 40 amino acid residues may be conveniently prepared by chemical synthesis or by enzymatic or chemical cleavage of the full-length or variant TSF receptor polypeptide. Other types of covalent modifications of the TSF receptor or fragments thereof are introduced into the molecule by reacting targeted amino acid residues of the TSF receptor or fragments thereof with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues.[0176]

Cysteinyl residues most commonly are reacted with α-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.[0177]

Histidyl residues are derivatized by reaction with diethyl-pyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1M sodium cacodylate at pH 6.0.[0178]

Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing α-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reaction with glyoxylate.[0179]

Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pK[0180]_aof the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.

The specific modification of tyrosyl residues may be made, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosyl residues are iodinated using[0181]¹²⁵I or¹³¹I to prepare labeled proteins for use in radioimmunoassay, the chloramine T method described above being suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R—N═C═N—R′), where R and R′ are different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.[0182]

Derivatization with bifunctional agents is useful for crosslinking TSF receptor to a water-insoluble support matrix or surface for use in the method for purifying a TSP member such as activin or inhibin or anti-TSF-receptor antibodies, and vice-versa. Commonly used crosslinking agents include, e.g., 1,[0183]₁-bis(diazo-acetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate), and bifunctional male imides such as bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively. These residues are deamidated under neutral or basic conditions. The deamidated form of these residues falls within the scope of this invention.[0184]

Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (T. E. Creighton,[0185]Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco, pp. 79-86 [1983]), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the TSF receptor polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide. By altering is meant deleting one or more carbohydrate moieties found in native TSF receptor, and/or adding one or more glycosylation sites that are not present in the native TSF receptor.[0186]

Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.[0187]

Addition of glycosylation sites to the TSF receptor polypeptide is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the native TSF receptor sequence (for O-linked glycosylation sites). For ease, the TSF receptor amino acid sequence is preferably altered through changes at the DNA level, particularly by mutating the DNA encoding the TSF receptor polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids. The DNA mutation(s) may be made using methods described above under the heading of “Amino Acid Sequence Variants of TSF Receptor Polypeptide.”[0188]

Another means of increasing the number of carbohydrate moieties on the TSF receptor polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. These procedures are advantageous in that they do not require production of the polypeptide in a host cell that has glycosylation capabilities for N- or O-linked glycosylation. Depending on the coupling mode used, the sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. These methods are described in WO 87/05330 published Sep. 11, 1987, and in Aplin and Wriston,[0189]CRC Crit. Rev. Biochem., pp.259-306 (1981).

Removal of carbohydrate moieties present on the TSF receptor polypeptide may be accomplished chemically or enzymatically. Chemical deglycosylation requires exposure of the polypeptide to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the polypeptide intact. Chemical deglycosylation is described by Hakimuddin, et al.,[0190]Arch. Biochem. Biophys.,259: 52 (1987) and by Edge et al.,Anal. Biochem.,118: 131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al.,Meth. Enzymol.,138: 350 (1987).

Glycosylation at potential glycosylation sites may be prevented by the use of the compound tunicamycin as described by Duskin et al.,[0191]J. Biol. Chem.,257: 3105 (1982). Tunicamycin blocks the formation of protein-N-glycoside linkages.

Another type of covalent modification of TSF receptor comprises linking the TSF receptor polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.[0192]

It will be appreciated that some screening of the recovered TSF receptor variant will be needed to select the optimal variant for binding to a TSF member and having the immunological and/or biological activity defined above. One can screen for stability in recombinant cell culture or in plasma (e.g., against proteolytic cleavage), high affinity to a TSF member, oxidative stability, ability to be secreted in elevated yields, and the like. For example, a change in the immunological character of the TSF receptor polypeptide, such as affinity for a given antibody, is measured by a competitive-type immunoassay. Other potential modifications of protein or polypeptide properties such as redox or thermal stability, hydrophobicity, or susceptibility to proteolytic degradation are assayed by methods well known in the art.[0193]

3. Uses of TSF Receptor Nucleic Acid, Polypeptide, and Antibodies Thereto[0194]

Nucleic acid encoding TSF receptor may be used as a diagnostic to determine the extent and rate of the expression of the TSF receptor in the cells of a patient. To accomplish such an assay, a sample of a patient's cells is treated, via in situ hybridization, or by other suitable means, and analyzed to determine whether the sample contains mRNA molecules capable of hybridizing with the nucleic acid molecule. Also, the TSF receptor nucleic acid can be used to detect nucleic acid encoding other TSF receptors. For example, such procedures as in situ hybridization, northern and Southern blotting, and PCR analysis may be used to determine whether DNA and/or RNA encoding a different TSF receptor is present in the cell type(s) being evaluated.[0195]

TSF receptor polypeptides are useful in radioreceptor assays to measure all bindable (presumably also active) forms of a TSF member such as inhibin or activin. If the TSF receptor is an inhibin receptor, such an assay would be specific for inhibin (i.e., activin would not be detected), and if the TSF receptor is an activin receptor, such an assay would be specific for activin (i.e., inhibin would not be detected). Such a radioreceptor assay would be conducted as described in the literature using the naturally purified or recombinant TSF receptor as the receptor element.[0196]

In addition, TSF receptor polypeptides are useful for screening for compounds that bind to them and have TSF biological activity as defined above. Preferably, these compounds are small molecules such as organic or peptide molecules that exhibit one or more of the desired activities of a TSF member such as inhibin or activin. Screening assays of this kind are conventional in the art, and any such screening procedure may be employed, whereby the test sample is contacted with the TSF receptor herein and the extent of binding and biological activity of the bound molecule are determined.[0197]

TSF receptor polypeptides are additionally useful in affinity purification of a TSF member or TSF-like molecules that bind to receptor (such as, for example, inhibin or activin or inhibin-like or activin-like molecules) and in purifying antibodies thereto. The receptor is typically coupled to an immobilized resin such as Affi-Gel 10 (Bio-Rad, Richmond, Calif.) or other such resins (support matrices) by means well known in the art. The resin is equilibrated in a buffer (such as one containing 25 mM TRIS, pH 7.5, 150 mM NaCl, and 0.1% OβG) and the preparation to be purified is placed in contact with the resin, whereby the molecules are selectively adsorbed to the receptor on the resin.[0198]

The resin is then sequentially washed with suitable buffers to remove non-adsorbed material, including unwanted contaminants, from the mixture to be purified, using, for an inhibin or activin mixture, e.g., 100 mM glycine, pH 3, and 0.1% octyl β glucoside. The resin is then treated so as to elute the TSF member compound using a buffer that will break the bond between the TSF member compound and TSF receptor (using, e.g., 100 mM glycine, pH 3, and 0.1% octyl β glucoside).[0199]

Therapeutic formulations of the receptor polypeptide for counteracting an excess amount of a TSF member are prepared for storage by mixing the receptor having the desired degree of purity with optional physiologically acceptable carriers, excipients, or stabilizers ([0200]Remington's Pharmaceutical Sciences, supra), in the form of lyophilized cake or aqueous solutions. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG).

The receptor polypeptide to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution. The receptor polypeptide ordinarily will be stored in lyophilized form or in solution.[0201]

Therapeutic TSF receptor polypeptide compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.[0202]

The route of TSF receptor or receptor antibody administration is in accord with known methods, e.g., injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, or intralesional routes, or by sustained release systems as noted below. TSF receptor is administered continuously by infusion or by bolus injection. TSF receptor antibody is administered in the same fashion, or by administration into the blood stream or lymph.[0203]

Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels [e.g., poly(2-hydroxyethyl-methacrylate) as described by Langer et al.,[0204]J. Biomed. Mater. Res.,15: 167-277 [1981] and Langer,Chem. Tech.,12: 98-105 [1982] or poly(vinylalcohol)], polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al.,Biopolymers,22: 547-556 [1983]), non-degradable ethylene-vinyl acetate (Langer et al., supra), degradable lactic acid-glycolic acid copolymers such as the Lupron Depot™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid (EP 133,988).

While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated proteins remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37° C., resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for protein stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.[0205]

Sustained-release TSF receptor compositions also include liposomally entrapped receptor. Liposomes containing receptor are prepared by methods known per se: DE 3,218,121; Epstein et al.,[0206]Proc. Natl. Acad. Sci. USA,82: 3688-3692 (1985); Hwang et al.,Proc. Natl. Acad. Sci. USA,77: 4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese patent application 83-118008; U.S. Pat. No. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. % cholesterol, the selected proportion being adjusted for the optimal receptor therapy.

An effective amount of TSF receptor to be employed therapeutically will depend, for example, upon the therapeutic objectives, the route of administration, and the condition of the patient. Accordingly, it will be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect. A typical daily human dosage might range from about 1 μg/kg to up to 100 mg/kg or more, preferably 0.01 mg/kg to 1 mg/kg, depending on the factors mentioned above. Typically, the clinician will administer the receptor polypeptide until a dosage is reached that achieves the desired effect in blocking excess TSF member. The progress of this therapy is easily monitored by conventional assays.[0207]

The TSF receptor molecules of the present invention may also be used to induce the formation of anti-TSF-receptor antibodies, which are identified by routine screening. Such antibodies may either be polyclonal or monoclonal antibodies, or antigen binding fragments of such antibodies (such as, for example, F(ab) or F(ab)[0208]₂fragments). Of particular significance to the invention are antibodies (and antigen-binding fragments of antibodies) that bind to any extracellular domain of the TSF receptor molecule. The most preferred anti-TSF-receptor antibodies (and antigen-binding fragments thereof) are those capable of preventing or inhibiting the binding of a TSF member to its receptor.

Polyclonal antibodies to the TSF receptor polypeptide generally are raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the TSF receptor polypeptide and an adjuvant. It may be useful to conjugate the TSF receptor polypeptide (including fragments containing the target amino acid sequence) to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl[0209]₂, or R¹N═C═NR, where R and R¹are different alkyl groups.

The route and schedule of the host animal or cultured antibody-producing cells therefrom are generally in keeping with established and conventional techniques for antibody stimulation and production. While mice are frequently employed as the test model, it is contemplated that any mammalian subject including human subjects or antibody-producing cells obtained therefrom can be manipulated according to the processes of this invention to serve as the basis for production of mammalian, including human, hybrid cell lines.[0210]

Animals are typically immunized against the immunogenic conjugates or derivatives by combining 1 mg or 1 μg of conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later the animals are boosted with {fraction (1/5)} to {fraction (1/10)} the original amount of conjugate in Freund's complete adjuvant (or other suitable adjuvant) by subcutaneous injection at multiple sites. Seven to 14 days later animals are bled and the serum is assayed for anti-TSF receptor polypeptide titer. Animals are boosted until the titer plateaus. Preferably, the animal is boosted with the conjugate of the same TSF receptor polypeptide, but conjugated to a different protein and/or through a different cross-linking agent. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are used to enhance the immune response.[0211]

Monoclonal antibodies are prepared by recovering immune cells—typically spleen cells or lymphocytes from lymph node tissue—from immunized animals and immortalizing the cells in conventional fashion, e.g., by fusion with myeloma cells or by Epstein-Barr (EB)-virus transformation and screening for clones expressing the desired antibody. The hybridoma technique described originally by Kohler and Milstein,[0212]Eur. J. Immunol.,6: 511 (1976) and also described by Hammerling et al., In:Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981) has been widely applied to produce hybrid cell lines that secrete high levels of monoclonal antibodies against many specific antigens.

It is possible to fuse cells of one species with another. However, it is preferable that the source of the immunized antibody-producing cells and the myeloma be from the same species.[0213]

The hybrid cell lines can be maintained in culture in vitro in cell culture media. The cell lines of this invention can be selected and/or maintained in a composition comprising the continuous cell line in hypoxanthine-aminopterin thymidine (HAT) medium. In fact, once the hybridoma cell line is established, it can be maintained on a variety of nutritionally adequate media. Moreover, the hybrid cell lines can be stored and preserved in any number of conventional ways, including freezing and storage under liquid nitrogen. Frozen cell lines can be revived and cultured indefinitely with resumed synthesis and secretion of monoclonal antibody.[0214]

The secreted antibody is recovered from tissue culture supernatant by conventional methods such as precipitation, ion exchange chromatography, affinity chromatography, or the like. The antibodies described herein are also recovered from hybridoma cell cultures by conventional methods for purification of IgG or IgM, as the case may be, that heretofore have been used to purify these immunoglobulins from pooled plasma, e.g., ethanol or polyethylene glycol precipitation procedures. The purified antibodies are sterile filtered, and optionally are conjugated to a detectable marker such as an enzyme or spin label for use in diagnostic assays of the TSF receptor in test samples.[0215]

While routinely mouse monoclonal antibodies are used, the invention is not so limited; in fact, human antibodies may be used and may prove to be preferable. Such antibodies can be obtained by using human hybridomas (Cote et al.,[0216]Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 [1985]). In fact, according to the invention, techniques developed for the production of chimeric antibodies (Morrison et al.,Proc. Natl. Acad. Sci.,81: 6851 [1984]; Neuberger et al.,Nature,312: 604 [1984]; Takeda et al.,Nature,314: 452 [1985]; EP 184,187; EP 171,496; EP 173,494; PCT WO 86/01533; Shaw et al.,J. Nat. Canc. Inst.,80: 1553-1559 [1988]; Morrison,Science,229: 1202-1207 [1985]; and Oi et al.,BioTechniques,4: 214 [1986]) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity (such as ability to activate human complement and mediate ADCC) can be used; such antibodies are within the scope of this invention.

Techniques for creating recombinant DNA versions of the antigen-binding regions of antibody molecules (known as Fab fragments), which bypass the generation of monoclonal antibodies, are encompassed within the practice of this invention. One extracts antibody-specific messenger RNA molecules from immune system cells taken from an immunized animal, transcribes these into complementary DNA (cDNA), and clones the cDNA into a bacterial expression system. One example of such a technique suitable for the practice of this invention was developed by researchers at Scripps/Stratagene, and incorporates a proprietary bacteriophage lambda vector system that contains a leader sequence that causes the expressed Fab protein to migrate to the periplasmic space (between the bacterial cell membrane and the cell wall) or to be secreted. One can rapidly generate and screen great numbers of functional Fab fragments for those that bind the antigen. Such TSF-receptor-binding molecules (Fab fragments with specificity for the TSF receptor polypeptide) are specifically encompassed within the term “antibody” as it is defined, discussed, and claimed herein.[0217]

In addition to their uses above, the TSF receptor polypeptides of the present invention are useful as the basis for assays of TSF member activity. Importantly, since such an assay measures a physiologically significant binding event, i.e., that of a hormone to its receptor, triggering a detectable change (such as phosphorylation, cleavage, chemical modification, etc.), it is likely to be both more sensitive and more accurate than immunoassays, which detect the physiologically non-significant binding of a TSF member to anti-TSF antibody. Moreover, the TSF receptor is capable of distinguishing its corresponding TSF member from other hormones with greater specificity than antibodies, which may cross-react with structurally similar molecules.[0218]

Although more sensitive and accurate than antibodies, the receptor molecules of the invention can be used to assay hormone levels in a sample in the same ways in which antibodies are used.[0219]

The anti-receptor antibodies of the present invention may also be used for diagnostic purposes, such as to measure the expression and function of a patient's TSF receptors. The anti-receptor antibodies also can be used in imaging to characterize tissue, or to define the presence and site of receptor-expressing cells.[0220]

For diagnostic purposes, the receptors and anti-receptor antibodies can be used in accordance with immunoassay technology. Examples of immunoassays are provided by Wide at pages 199-206 of[0221]Radioimmune Assay Method, Kirkham and Huner, ed., E & S. Livingstone, Edinburgh, 1970.

Thus, in one embodiment, TSF receptor molecules can be detectably labeled and incubated with a test sample containing TSF-member-like molecules (such as biological fluids, e.g., serum, sputum, urine, etc.), and the amount of receptor molecule bound to the sample is ascertained. In a second embodiment, antibody to the receptor, or to a TSF member, can be used to create a “pseudo-sandwich immunoassay.” In one such assay (a “forward” assay) a sample suspected of containing a TSF member can be incubated in the presence of an immobilized anti-TSF antibody. Solubilized, detectably labeled TSF receptor molecules are added to the reaction mixture, and the amount of TSF member is determined by measuring the amount of bound receptor.[0222]

As will be evident to those of ordinary skill, various alternative assays can also be devised. The assay may be a simple “yes/no” assay to determine whether a TSF member is present or may be made quantitative by comparing the measure of labeled molecule with that obtained for a standard sample containing known quantities of the TSF member.[0223]

In another diagnostic test suitable for the receptor herein, “simultaneous” and “reverse” assays are used. A simultaneous assay involves a single incubation step as the antibody (or receptor) bound to the solid support and labeled receptor (or antibody) are both added to the sample being tested at the same time. After the incubation is completed, the solid support is washed to remove the residue of fluid sample and uncomplexed labeled antibody. The presence of labeled molecules associated with the solid support is then determined as it would be in a conventional sandwich assay.[0224]

In the “reverse” assay, stepwise addition first of a solution of labeled molecule (either receptor or antibody) to the fluid sample followed by the addition of unlabeled molecule (either antibody or receptor) bound to a solid support after a suitable incubation period is utilized. After a second incubation, the solid phase is washed in conventional fashion to free it of the residue of the sample being tested and the solution of unreacted labeled antibody. The determination of labeled antibody associated with a solid support is then determined as in the simultaneous forward assays.[0225]

The assay of this type requires that at least one binding partner be labeled with a reporter molecule. Examples of types of labels that can be used in the present invention include, but are not limited to, enzyme labels, radioisotopic labels, non-radioactive isotopic labels, fluorescent labels, toxin labels, and chemiluminescent labels.[0226]

Examples of suitable enzyme labels include malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast-alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, e.g., horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, acetylcholine esterase, etc.[0227]

Examples of suitable radioisotopic labels include[0228]¹¹¹In,³²P,¹⁴C,¹²⁵I,³H,¹³¹I,³⁵S,⁵¹Cr,⁵⁷To,⁵⁸Co,⁵⁹Fe,⁷⁵Se,¹⁵²Eu,⁹⁰Y,⁶⁷Cu,²¹⁷Ci,²¹¹At,²¹²Pb,⁴⁷Sc,¹⁰⁹Pd, and the like. Examples of suitable non-radioisotopic labels include¹⁵⁷Gd, 55Mn,⁵²Tr,⁵⁶Fe, etc.

Examples of suitable fluorescent labels include an[0229]¹⁵²Eu label, a fluorescein label, an isothiocyanate label, a rhodamine label, a phycoerythrin label, a phycocyanin label, an allophycocyanin label, an o-phthaladehyde label, a fluorescamine label, etc.

Examples of suitable toxin labels include diphtheria toxin, ricin, and cholera toxin. Examples of chemiluminscent labels include a luminal label, an isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label, a luciferase label, an aequorin label, and the like.[0230]

Those of ordinary skill in the art will know of other suitable labels that may be employed in accordance with the present invention. The binding of these labels to receptors, antibodies, or fragments thereof can be accomplished using standard techniques commonly known to those of ordinary skill in the art. For instance, coupling agents such as dialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotized benzidine, and the like may be used to tag the polypeptide with the above-described fluorescent, chemiluminescent, and enzyme labels. See, for example, U.S. Pat. Nos. 3,940,475 (fluorimetry) and 3,645,090 (enzymes); Hunter et al.,[0231]Nature,144: 945 (1962); David et al.,Biochemistry,13: 1014-1021 (1974); Pain et al.,J. Immunol. Methods,40: 219-230 (1981); Nygren,J. Histochem. and Cytochem.,30: 407-412 (1982); O'Sullivan et al., “Methods for the Preparation of Enzyme-antibody Conjugates for Use in Enzyme Immunoassay,” inMethods in Enzymology, ed. J. J. Langone and H. Van Vunakis, Vol. 73 (Academic Press, New York, N.Y., 1981), pp. 147-166; Kennedy et al.,Clin. Chim. Acta,70: 1-31 (1976); and Schurs et al.,Clin. Chim. Acta,81: 1-40 (1977). Coupling techniques mentioned in the lattermost reference are the glutaraldehyde method, the periodate method, the dimaleimide method, and the m-maleimidobenzyl-N-hydroxysuccinimide ester method.

In the practice of the present invention, enzyme labels are a preferred embodiment. No single enzyme is ideal for use as a label in every conceivable assay. Instead, one must determine which enzyme is suitable for a particular assay system. Criteria important for the choice of enzymes are turnover number of the pure enzyme (the number of substrate molecules converted to product per enzyme site per unit of time), purity of the enzyme preparation, sensitivity of detection of its product, ease and speed of detection of the enzyme reaction, absence of interfering factors or of enzyme-like activity in the test fluid, stability of the enzyme and its conjugate, availability and cost of the enzyme and its conjugate, and the like. Included among the enzymes used as preferred labels in the assays of the present invention are alkaline phosphatase, horseradish peroxidase, beta-galactosidase, urease, glucose oxidase, glucoamylase, malate dehydrogenase, and glucose-6-phosphate dehydrogenase. Urease is among the more preferred enzyme labels, particularly because of chromogenic pH indicators that make its activity readily visible to the naked eye.[0232]

Immobilization of reagents is required for certain assay methods. Immobilization entails separating the TSF receptor from any analyte that remains free in solution. This conventionally is accomplished by either insolubilizing the receptor before the assay procedure, as by adsorption to a water-insoluble matrix or surface (Bennich et al., U.S. Pat. No. 3,720,760), by covalent coupling (for example, using glutaraldehyde cross-linking), or by insolubilizing the molecule afterward, e.g., by immunoprecipitation.[0233]

The foregoing are merely exemplary diagnostic assays for TSF members. Other methods now or hereafter developed for the determination of these analytes are included within the scope hereof.[0234]

The following examples are offered by way of illustration and not by way of limitation. The disclosures of all literature references cited in the specification are expressly incorporated herein by reference.[0235]

EXAMPLE IPurification of Bovine Inhibin/Activin Receptor

A. General Membrane Extract (ME) Procedure[0236]

Bovine pituitaries (in an amount of 200 g unless otherwise indicated) were suspended in 150 mM NaCl (latter ME had protease inhibitor cocktail added, which comprised 1 mM EDTA, 25 g/ml aprotinin, 0.5 μg/ml leupeptin, and 0.7 μg/ml pepstatin), disrupted in a blender, and subsequently mixed with a stir bar at 5° C. for one hour. The mixture was then sedimented at 10,000×g to remove particulate matter. The resulting supernatant was centrifuged at 105,000×g for 3 hours. The ultracentrifugation pellet was resuspended in 85 mM TRIS, pH 7.8, 0.1% octyl β glucoside (OβG), 30 mM NaCl, 1 mM EDTA, and 25 μg/ml aprotinin.[0237]

Several MEs were prepared separately by this procedure, numbered in the order of their preparation from ME1 to ME9. For ME preparations 3-9, the supernatant was treated with DNase (Boehringer Mannheim) overnight at 1 unit/μg DNA to reduce viscosity and improve sedimentation of particulate matter during ultracentrifugation. DNase-treated samples were sedimented at 105,000×g for 3 hours and clear red supernatants were found. For ME preparations 4-9, 0.5 μg/ml leupeptin and 0.7 μg/ml pepstatin were also added. The resulting suspension was sedimented at 105,000×g.[0238]

B. FITC-Conjugated Inhibin[0239]

The pH of recombinant human inhibin (Mason, U.S. Pat. No. 4,798,885, supra) was adjusted to 8.0 with 1 M Na[0240]₂CO₃, pH 9.3. A total of 7 mg of fluorescein isothiocyanate (FITC, Sigma) was added to 140 μl dimethyl formamide (DMF) and subsequently diluted in water to a final concentration of 1 μg/μl. A total of 2 μg of this FITC solution was added to 10 μg of the inhibin to give a total volume of 100 μl. After exactly one hour of end-over-end mixing, the sample was sedimented in a microcentrifuge to remove particulate matter (14,000 rpm for 15 min.). Free FITC was removed from the sample by diafiltration using a 10-Kd molecular weight cut-off ultrafiltration (UF) membrane (Ultrafree-MC Filter Unit, Millipore). Conjugated inhibin was used on the day of preparation. If the analysis was to be carried over to the following day, the conjugated hormone was stored at −70° C.

C. Size-Exclusion Chromatography of ME Incubated With FITC-Conjugated Inhibin[0241]

A Perkin-Elmer fluorescence detector was connected to a Hewlett Packard HP 1090 HPLC ([0242]excitation 480 nm, emission 525 nm). A chart recorder or an integrator was used to monitor the fluorescent peaks. 50 μl of ME1 was added to 140 μl of elution buffer (100 mM P04, pH 6.0±0.1, and 0.1% OβG) with 5-10 μl of inhibin-FITC (assuming 100% recovery of FITC-labeled inhibin; [hormone]=0.7 μg/μl). After a 30-min. incubation at ambient temperature, 50 μl of sample was loaded. The TSK 3000 SWXL size-exclusion column with guard column was eluted with the elution buffer described above using a flow rate of 1 ml/min.

Inhibin-FITC eluted at a molecular weight less than expected (27 Kd vs. 32 Kd) relative to the gel filtration standards (Sigma), suggesting some interaction with the TSK gel. This observation was also seen with unlabeled inhibin, indicating that the FITC tag was not responsible for the delay in retention time. A fluorescent peak was observed at a molecular weight greater than 200 Kd (i.e., near the void), suggesting that there was a protein(s) in the ME that bound to inhibin-FITC. Fluorescent peaks were collected and analyzed using SDS-PAGE. Many proteins co-eluted in the 200 Kd+peak. Furthermore, there was no detectable inhibin-FITC band observed on the gels (maximum recoverable inhibin-FITC=0.7 μg/μl×10 μl=7 μg).[0243]

D. SDS-PAGE Analysis[0244]

For all SDS-PAGE analyses conducted the following conditions were employed unless otherwise indicated. Ten percent acrylamide SDS-PAGE was used. If the PAGE was reduced, the reducing agent was 5% β-mercaptoethanol. The percent SDS of the sample buffer was 2%. The molecular weight markers employed were myosin (200,000),[0245]E. colip-galactosidase (116,250), rabbit muscle phosphorylase b (97,400), bovine serum albumin (66,200), and hen egg white ovalbumin (42,699). The molecular weights were determined by plotting log molecular weight versus molecular weight marker migration. Silver staining was employed except when indicated otherwise.

E. Ligand Affinity Chromatography Columns in General[0246]

Four support matrices were utilized to immobilize a biologically active (in the rat pituitary FSH-release assay) form of recombinant inhibin A (as described by Mason, U.S. Pat. No. 4,798,885, supra) that was proteolytically clipped at amino acid 14 to 18 during production and/or isolation. All resins utilized a spacer arm to prevent steric hindrance from interfering with the isolation of receptor. Inhibin A in[0247]^˜30% acetonitrile, 0.1% trifluoroacetic acid, pH^˜3, was concentrated from^˜0.1 to 1 mg/ml using a stirred cell withYM 10 UF membrane (Amicon). The pH was rapidly increased to 5 or 8, depending on the support resin, with 1 M citrate, pH 5 or 1 M TRIS, pH 8, respectively. Subsequently, inhibin A was buffer exchanged into coupling buffer (0.1 M citrate buffer, pH 5, or 0.1 M sodium bicarbonate, pH 8) according to manufacturer's protocol usinggel filtration PD 10 columns (Pharmacia).

1.[0248]Affi Prep 10 Support Matrix

About 9 mg inhibin A was immobilized to 3 ml of[0249]Affi Prep 10 support matrix as recommended by its manufacturer, BioRad. This resin was used once before switching toAffi Gel 10. Affi Prep 10 (hydrophilic polymer) and Affi Gel 10 (crosslinked agarose) share the same spacer arm (10 atoms) and coupling chemistry (N-hydroxysuccinimide displaced>stable amide bond formed) through the free amino groups of the inhibin.

2.[0250]Affi Gel 10

Two batches of inhibin A coupled to[0251]Affi Gel 10 were prepared as directed by BioRad (9 mg inhibin coupled to 3 ml Affi Gel per batch, carbonate coupling buffer). TRIS was also immobilized to Affi Gel to prepare precolumns for reducing non-specific adsorption to the affinity column support. Five mg of human recombinant activin A (Mason, U.S. Pat. No. 4,798,885, supra,) was also immobilized to^˜3 ml ofAffi Gel 10.

3. Affi Gel HZ[0252]

Affi Gel HZ was used to immobilize inhibin A through the carbohydrate groups of the alpha chain. The procedure described by BioRad was used to couple the ligand to the support.[0253]

4. HiPAC[0254]

A silica-based support with activated aldehyde groups was used to immobilize inhibin A through the primary amino groups (ChromatoChem).[0255]

F. Isolation of Inhibin/Activin Receptor Using Ligand Affinity Chromatography[0256]

1. Ligand Affinity Chromatography 1[0257]

ME2 (cloudy) was loaded on an Affi Prep/inhibin column. The affinity column was extensively washed with 10 mM TRIS, pH 7.5, and 0.1% OβG. After a stable A[0258]₂₈₀baseline was achieved, proteins were eluted from the affinity column with 100 mM acetic acid, pH 3, and 0.1% OβG. Eluent was immediately neutralized with 3 M TRIS, pH 8.5, and stored at 5° C.

SDS-PAGE Analysis[0259]

SDS-PAGE analysis revealed that the eluent contained ligand as well as many other proteins. Consequently, direct sequencing of the eluent was difficult.[0260]

N-Terminal Sequence Analysis[0261]

The strongest amino acid sequence signal found for a protein bound to a 2-cm, 15-μm in diameter C4 reverse phase column was that of the β chain of hemoglobin.[0262]

Three significant changes were subsequently made in the ligand affinity column procedures. Further recoveries used[0263]Affi Gel 10 as the support gel (a frequently used resin in several papers describing the isolation of receptors). In addition, a more stringent wash buffer with 150 mM NaCl as well as a precolumn were incorporated into the procedure to reduce non-specific protein binding.

2. Ligand Affinity Chromatography 2[0264]

ME3 was loaded on an Affi Gel-Tris precolumn (BV=1 ml) and subsequently through an Affi Gel-inhibin affinity column (BV=3 ml, 3 mg inhibin/ml resin, run in series after the precolumn). The affinity column was extensively washed with 25 mM TRIS, pH 7.5, 150 mM NaCl, and 0.1% OβG. The putative receptor was then eluted with 100 mM glycine, pH 3, and 0.1% OβG. Eluent (˜5 ml) was immediately neutralized with 1 M NaOH and stored at −20° C. Incubation of FITC-labeled inhibin with the flow-through ME fraction and subsequent size-exclusion chromatography analysis indicated that there was potentially more receptor remaining. As a result, the flow-through fraction was diluted {fraction (1/4)} with 100 MM PO[0265]₄, pH 6, and 0.1% OβG (final volume=9.5 ml), filtered and recirculated for 45 min. (flow rate=3 ml/min.) over the inhibin column. After a 40-min. wash, the column was eluted (^˜5 ml). SDS-PAGE indicated that the proteins found in the initial purification were identical to those recovered from the flow-through.

SDS-PAGE Analysis of Flow-Through Fraction Eluent[0266]

The eluent contained three major proteins as determined by a non-reduced SDS-PAGE gel. The molecular weights of the three proteins were approximately 32 Kd (inhibin from ligand affinity column), 60 Kd, and over 200 Kd. Upon reduction with β-mercaptoethanol, the 200 Kd+ protein band migrated to[0267]^˜150 Kd.

N-Terminal Sequence Analysis[0268]

Flow-through fraction eluent was loaded on a C4 reverse phase HPLC column (15 μm bead diameter,[0269]column height 20 mm, n-propanol/TFA linear gradient). Peaks at approximately 11, 19, and 23 min. were determined to be an artifact, inhibin, and the potential receptor, respectively, by reduced SDS-PAGE. No detectable amino acids were found in the sequencing of the material remaining on the C4 column, suggesting that all of the proteins had been eluted. The background of the sequencing chromatograms was stable with successive cycles, suggesting that there were no N-terminal blocked proteins on the C4 column. The C4-isolated potential receptor did not yield an amino acid sequence, suggesting that there was insufficient protein for detection.

3. Ligand Affinity Chromatography 3[0270]

To recover more receptor, 500 instead of 200 g of bovine pituitaries was prepared for ME4. (Unless noted otherwise, all subsequent MEs were derived from 500 g of pituitaries.) ME4 was loaded on an Affi Gel-10.TRIS column (bed volume (BV)=1 ml), an inhibin A column immobilized through the carbohydrate groups (Affi Gel-HZ, 3-ml BV, 9 mg inhibin), and finally through an inhibin A column immobilized through the free amino groups (Affi Gel-10, 3-ml BV, 9 mg inhibin). The affinity columns were extensively washed with 25 mM TRIS, pH 7.5, 150 mM NaCl, and 0.1% OβG. The putative receptor was then eluted with 100 mM glycine, pH 3, and 0.1% OβG from both inhibin columns separately. The eluents ([0271]^˜5 ml) were immediately neutralized with 3 M TRIS, pH 8. Only^˜25 of the 45 ml of ME4 was passed through the columns because of particulate matter fouling the precolumn. Upon further investigation, it was found that ME4 was viscous due to DNA that retarded the sedimentation of particles. The remaining 20 ml was treated with DNase according to manufacturer's instructions at 5° C. (Boehringer Mannheim). The solution was diluted with wash buffer to 35 ml, centrifuged at 105,000×g for 1.5 hours, and then loaded on the columns (new precolumn). No plugging of the precolumn was observed. [There were large pellets at the bottom of the ultracentrifugation tubes after DNase treatment.] The 35-ml sample was recirculated through the columns for 30 minutes. After a 60-minute wash, the columns were eluted separately. The eluents were immediately neutralized and stored at −20° C.

SDS-PAGE Analysis[0272]

Using reduced SDS-PAGE, it was determined that the 150-Kd protein thought to be the inhibin receptor was present in the Affi Gel-10 inhibin eluent, but it was absent in the Affi Gel HZ-inhibin eluent. This result suggests that the orientation of inhibin immobilized through the carbohydrate group prevented the binding of the putative receptor to the ligand. The high-molecular-weight protein thought to be the inhibin receptor apparently did not elute from the reverse phase C4 column at the same retention time as was observed with ME3. This might be the result of the additional protease inhibitors added to ME4, the use of a different batch of bovine pituitaries, or the storage of the sample at −20° C. immediately after elution.[0273]

N-Terminal Sequence Analysis[0274]

The N-terminal sequence of a protein bound to the C4 column was valine connected to leucine connected to serine, histidine, or cysteine, followed by glutamic acid. This sequence was observed for both the DNase-treated and untreated ME. Over fifty proteins were found in the Dayhoff protein database for each of the three potential 4-amino-acid N-terminal sequences (i.e., 4 amino acids are insufficient for protein identification).[0275]

4. Ligand Affinity Chromatography 4[0276]

ME5 was passed through an Affi Gel-10-TRIS column (BV=2 ml), and two inhibin columns connected in series (3-ml BV/each, started at 9 mg inhibin/each). Columns were extensively washed with 25 mM TRIS, pH 7.5, 150 mM NaCl, and 0.1% OβG. The putative receptor was then eluted with 100 mM glycine, pH 3, and 0.1% OβG. The flow-through fraction as well as the residual supernatant that was remaining after the final ultracentrifugation ([0277]^˜5 ml of sample that was cloudy in appearance, so it was not incorporated into the first ME sample) was reprocessed over the columns. A final third reprocessing recovery was also performed. All eluents were immediately neutralized with 3 M TRIS, pH 8, and stored at −20° C.

SDS-PAGE and N-Terminal Sequence Analysis[0278]

Using RP-HPLC (15-μm bead diameter C4 resin in a 2-cm column, 0-70% n-propanol/TFA gradient) the receptor was concentrated and purified to homogeneity. The receptor-enriched RP fractions were run on reduced 10% SDS-PAGE, transferred to PVDF, and stained with Coomassie blue.[0279]

The single protein band at about 150 Kd believed to be the inhibin receptor was cut out of the transblot and sequenced. The N-terminal sequence was found to be:[0280]

ValLeuThrGluGluThrGluIleIleMetProThrProLysProGluLeuXaaAlaXaaXaaAsn (Sequence Identity No. 1).[0281]

There is a proteolytic clip site between the initial ValLeuThr and the remainder of the sequence. The Xaa residues indicate unknown amino acids. The clip site provided conformation of the amino acids in the sequence (i.e., the same amino acid came up three cycles later).[0282]

5. Ligand Affinity Chromatography 5[0283]

ME6 was passed through a TRIS column (Affi Gel-10, BV=1 ml) and an inhibin column (Affi Gel-10, 6-ml BV, started at 18 mg inhibin). The affinity column was thoroughly washed (45 BV) using a wash buffer of 25 mM TRIS, pH 7.5, 150 mM NaCl, and 0.1% OβG. The putative receptor was acid eluted with 100 mM glycine, pH 3, and 0.1% OβG. The flow-through fraction was reprocessed over the column. All eluents were immediately neutralized with 1 M TRIS, pH 8, and stored at −20° C.[0284]

SDS-PAGE and N-Terminal Sequence Analysis[0285]

Ten percent of the eluents were used to determine the recovery yield of RP-HPLC ([0286]^˜40% recovery). Seventy percent was used to confirm the sequence of the 150-Kd protein by running on reduced SDS-PAGE, transferring to PVDF, and staining with Coomassie blue. The sequence for dimerized bovine serum albumin was found at^˜150 Kd on the transblot from the reduced SDS gel. Unlike before, there was no RP-HPLC step to enrich for the putative receptor. Instead, the eluent sample was dialyzed against 10 mM TRIS, pH 7.5 and 0.1% OβG, dried down, run on SDS-PAGE, and transblotted to PVDF. This suggests that the N-terminus of the inhibin receptor was blocked, that the RP-HPLC step is required to obtain sequence, or that the wrong band was cut out of the PVDF (there did appear to be another faint band just above the one that was cut out).

6. Ligand Affinity Chromatography 6[0287]

150 g of bovine pituitaries were used in preparing ME7. The 25 ml of ME was diluted to 40 ml with wash buffer. The diluted ME was divided into 4×10-ml samples. Ten ml of ME was loaded on the affinity column (BV=6 ml). The column was washed for 1.5 hours with 25 mM TRIS, pH 7.5, 150 mM NaCl, and 0.1% OβG (flow rate=3 ml/min). The putative receptor was then eluted with 100 mM glycine, pH 3, and 0.1% OβG. The eluent was immediately neutralized with 3 M TRIS. The flow-through fraction was also reloaded, and treated as above. The TRIS precolumn from this procedure was also washed and eluted for gel analysis.[0288]

A 50-ml sample of 0.1 mg/ml inhibin was concentrated to[0289]^˜2 ml using a YM-10 ultrafiltration membrane in an Amicon stirred cell. The concentrated sample was neutralized with 1 M TRIS, pH 8 (^˜100 μl) and added to 10 ml of ME. After a 3-hour incubation, the ME was loaded on the affinity column. The wash and elution conditions were identical to what was described above (new TRIS precolumn was used). The flow-through fraction was also loaded, and treated as above.

Ten ml of ME7 was extensively dialyzed against the wash buffer and then acidified to pH 3. Subsequently, the ME7 was filtered through a 100-Kd MWCO membrane (Ultrafree-MC Filter Unit, Millipore). The filtrate was assayed for inhibin and activin in the respective ELISAs.[0290]

Observations[0291]

The addition of free inhibin into the ME prior to loading on the immobilized inhibin column greatly reduced the recovery of the putative receptor.[0292]

No detectable inhibin (<0.7 ng/ml) or activin (<0.5 ng/ml) was measured in the acid-treated ME passed through a 100-Kd ultrafiltration membrane, suggesting that the inhibin observed in the eluent results from the affinity column and not from the ME.[0293]

7. Ligand Affinity Chromatography 7[0294]

ME8 (125-g preparation from bovine pituitaries) was loaded on a TRIS column (BV=1 ml), an inhibin column (Affi Gel-10, 6-ml BV, started at 18 mg inhibin), and finally an activin column (Affi Gel-10, 3-ml BV, 5 mg activin). Affinity columns were extensively washed for 90 minutes at a flow rate of 3 ml/min with 25 mM TRIS, pH 7.5, 150 mM NaCl, and 0.1% OβG. Columns were individually eluted with 100 mM glycine, pH 3, and 0.1% OβG. The flow-through fraction was reprocessed. All eluents were immediately neutralized with 1 M TRIS, pH 7.5, and stored at −20° C.[0295]

The remaining ME8 (125-g preparation of bovine pituitaries) was used to evaluate inhibin immobilized to a silica-based HPLC column (ChromatoChem).[0296]

HPLC Time Table

	Time (min.)	% A	% B	Flow Rate (ml/min.)

0	100	0	2
25	0	100	2
25.01	100	0	2
35	100	0	2

Buffer A: 10 mM PO[0297]₄, pH 6.8, 100 mM Na₂SO₄and 0.1% OβG.

Buffer B: 10 mM PO[0298]₄, pH 2.5, 100 mM Na₂SO₄and 0.1% OβG.

2 ml of ME8 was loaded onto the 0.83-ml BV column per run (n=2).[0299]

1-ml fractions were collected throughout the isolation procedure.[0300]

Fractions that had a pH<5 were characterized by SDS-PAGE.[0301]

SDS-PAGE Analysis[0302]

The eluents from the immobilized activin and inhibin columns both had a single protein band at 135-150 Kd, depending on the size of the gel, by silver stain on reduced SDS-PAGE. The molecular weight was 135 Kd using a 8×10 cm gel and was 148 Kd using a 12×15 cm gel.[0303]

N-Terminal Sequence Analysis[0304]

For the Affi Gel columns, more putative receptor protein was found in the activin eluent that in the inhibin column eluent. N-terminal sequencing of the 135-150-Kd band on transblots revealed that the first 15-20 amino acids were identical for the inhibin and activin column eluents. These data also confirmed the previous sequence observed from Ligand Affinity Chromatography 4. Twenty percent of the inhibin eluent was clipped at the valine-leucine-threonine and glutamic acid-glutamic acid juncture and 50% of the activin column eluent was clipped at the same position. The RP-HPLC profiles for the inhibin and activin column eluents were also similar.[0305]

In addition to the Dayhoff protein database that suggested that the 135-150-Kd band was an unknown protein, a larger Genbank DNA database also indicated a novel sequence. Another major protein band at 80 Kd (reduced SDS-PAGE) was found to have the sequence:[0306]

AlaValAsnProThrGlyArgAspAlaValGluProValAlaValXaa(Ala)LeuAspLeuIleAsnLys (Sequence Identity No. 2),[0307]

where Xaa is an unknown amino acid and the Ala in parenthesis is believed to be correct. This sequence was found also to be absent from the databases. The protein was not affected in the competition studies, suggesting some non-specific interaction with columns.[0308]

The activin column eluent that had been transferred to PVDF and sequenced for[0309]^˜20 cycles was digested with cyanogen bromide. The resulting digest was sequenced and 3-5 peptide sequences were observed, suggesting either that the number of methionines was low or that the protein was highly glycosylated. The sequence occurring after the methionine atposition 10 was not found, indicating that the N-terminus for the activin receptor was not blocked.

The inhibin-HiPAC column was found to improve greatly the receptor recovery yield based on SDS-PAGE while reducing purification time from −2.5 hours to 35 mins. The 80-Kd protein was also observed in the inhibin-HiPAC eluent. The eluent fractions from the HPLC column were those used to confirm the N-terminal sequence of the inhibin receptor (no RP-HPLC step prior to sequencing).[0310]

8. Ligand Affinity Chromatography 8[0311]

ME9 (equivalent to 125 g of bovine pituitaries) was passed through a TRIS column and an activin column. Columns were extensively washed (45 BV) with 25 mM TRIS, pH 7.5, 150 mM NaCl, and 0.1% OβG. Putative activin receptor was then eluted with 100 mM glycine, pH 3, and 0.1% OβG. The flow-through fraction was reprocessed over the column. All eluents were immediately neutralized with 1 M TRIS, pH 7.5, and stored at −20° C.[0312]

The remaining ME (125 g bovine pituitaries) was divided into four fractions: a control, a 60-70° C. heat denaturation (not examined since protein precipitation formed in[0313]^˜15 min.), a 2-mg inhibin spike-back, and a 100-Kd ultrafiltration/diafiltration fraction (i.e., 5 ml of ME diluted into 250 ml of low-pressure wash buffer and concentrated to^˜5 ml with 100 Kd Amicon UF membrane). These so-treated ME mixtures were purified using the inhibin-HiPAC HPLC system.

HPLC Time Table

Time (min.)	% A	% B	Flow Rate (mi/min.)

0	100	0	1 at 5 min. 2 ml/min.
20	100	0	2
25	0	100	2
25.01	100	0	2
35	100	0	2

Analysis[0314]

Activin-Affi Gel Column[0315]

Most of the activin column eluents, both initial and reprocessed, were used to determine internal sequence.[0316]

Inhibin-HiPAC Column[0317]

There were few proteins in the wash buffer just prior to the elution of putative inhibin receptor. A majority of the putative receptor was eluted in the first 5 BV of elution buffer. Reprocessing the flow-through fraction in this system yielded less receptor than what had been observed using the inhibin-Affi Gel column. That is, the HiPAC column even with less ligand bound was more efficient in recovering the alleged receptor than the Affi Gel column. The UF/DF procedure to reduce the number of contaminating proteins in the eluent was not effective and in fact reduced the recovery of the inhibin receptor. The addition of 2 mg inhibin to the ME greatly reduced the recovery of the 135-150-Kd protein (reduced SDS-PAGE).[0318]

G. Cyanogen Bromide Digestion of Activin Receptor for Determining Internal Sequence[0319]

The activin column eluent from ligand affinity chromatography 8 described above was subjected to analysis by reduced SDS-PAGE under the conditions described above. The gel results were that the major protein in the eluent was the 135-150-Kd protein, which is the putative receptor. As a result, the major sequence observed after the cyanogen bromide digest should be that of the receptor. The activin column eluent was treated with dithiothreitol for one hour at 40° C. to reduce oxidized methionine (oxidized methionine is not cleaved by cyanogen bromide). The reduced sample was then incubated with cyanogen bromide overnight at room temperature using standard procedures as described, e.g., in Spande et al.,[0320]Adv. Protein Chem.,24: 97 (1970) and Gross and Witkop,J. Biol. Chem.,237: 1856 (1962). The peptides resulting from the digest were separated using RP-HPLC (2-cm C4 column) with a propanol/TFA linear gradient (0-70%). The amino acid sequence of the peptides in the RP-HPLC fractions was determined.

Two potential internal sequences were identified:[0321]

1) MetTyrAlaProGluTyr (Sequence Identity No. 3)[0322]

2) MetIlePheAlaLeuLeuPhePheGly (Sequence Identity No. 4)[0323]

H. Rat Germ Cell Competition Assay[0324]

Inhibin A and activin A as described above with and without FITC label were diluted in an elution buffer of 100 mM phosphate, pH 6.0, 0.1% (w/v) SDS in the presence and absence of 21 mM dithiothreitol, and subsequently heated to 10° C. for 5 min. Approximately 100 ng of denatured hormone was injected on a TSK 3000 SWXL column using an HP 1090 high-performance liquid chromatography system (Hewlett Packard). The column was eluted with the same elution buffer using a flow rate of 0.5 ml/min. Peaks were monitored with a fluorescence detector ([0325]excitation 480 nm, emission 525 nm). Molecular weights were determined for the hormones by plotting log molecular weight for the gel filtration standards (Sigma): albumin (66 Kd), carbonic anhydrase (29 Kd), and cytochrome C (12.4 Kd) as a function of elution volume/void volume (Ve/Vo).

The size-exclusion chromatography is shown in FIG. 2. No autofluorescence was observed for the unlabeled proteins. For the FITC-conjugated proteins, only mature inhibin and activin peaks were present, indicating the absence of protein contaminants, hormone precursors, and SDS-stable aggregates. Calculated molecular weights for inhibin A and activin A were 27 Kd (actual 32 Kd) and 28 Kd (actual 28 Kd), respectively. Upon reduction with dithiothreitol (DTT), the elution of the inhibin α chain (18 Kd) was found to be retarded relative to the β[0326]_Achain (14 Kd), suggesting that the discrepancy in the actual and calculated molecular weights for inhibin resulted from the α chain adsorbing to the size-exclusion gel. Peak areas for the α and β chains of inhibin suggest a FITC conjugation ratio of 1:2 (maximum theoretical ratio 2:9).

FITC-labeling of inhibin, activin, and ovalbumin (background) was performed as described by Woodruff et al., in[0327]Methods in Enzymology: Growth Factors, part C, Barnes, D., Mather, J., Sato, G. (eds), Academic Press, San Diego, Calif., 198: 347-356 (1991), with the modifications as described by Chatelier et al.,EMBO J.,5: 1181-1186 (1986). Briefly, 10 μg of inhibin A, activin A, activin B, or ovalbumin was mixed with 1 μg FITC and allowed to react for one hour at room temperature. Unconjugated FITC was removed from the reaction by diafiltration. Conjugated hormone was analyzed by size-exclusion chromatography. Bioactivity was measured using a rat pituitary bioassay.

Thirty-one-day-old male Sprague-Dawley rats (Charles Rivers Laboratories, Wilmington, Mass.) were sacrificed by carbon dioxide asphyxiation. The testes of these rats were removed and placed directly into DMEM:F12 (1:1) medium with HEPES and gentamycin (50 μg/ml). The testes were decapsulated and the interstitial tissue was lysed with solution A [1 M glycine, 2 mM EDTA, 20 IU/ml DNase I (Boehringer-Mannheim) and 0.01% soy trypsin inhibitor (STI) (Gibco) in Ca[0328]⁺²Mg⁺²-free PBS, pH 7.2] and the tissue was exposed briefly (5-10 min) to collagenase-dispase [in Dulbecco's Modified Eagle's/Ham's F12 nutrient mixture (1:1 v/v) (Gibco) supplemented with 1.2 g/l sodium bicarbonate, 10 mM HEPES buffer, 20 mg/l gentamycin sulfate, and 1 mg/ml bovine serum albumin]. Tubules were minced with scissors to 1-3 mm pieces and passed 3-4 times through an 18-gauge needle using a sterile syringe. The suspension was washed by centrifugation and resuspended in fresh medium. Clumps of Sertoli cells were trapped in a 20-μM Nytex™ nylon mesh with the single cells (germ cells) passing through. Germ cells were freed of debris by repeated washing (4-6 times) in medium. The enriched germ cell preparation was cultured overnight in primary testes cell medium [15 mM Dulbecco's Modified Eagle's/Ham's F12 nutrient mixture (1:1 v/v) (Gibco) supplemented with 1.2 g/l sodium bicarbonate, 10 mM HEPES buffer, 20 mg/l gentamycin sulfate, 5 μg/ml insulin (Sigma, St. Louis, Mo.), 5 ng/ml human transferrin (Sigma), 5 ng/ml epidermal growth factor (Sigma), 5 ng/ml α-tocopherol (Sigma), and 25 μg/ml aprotinin (Miles Laboratories, Naperville, Ill.)] plus Sertoli cell-conditioned medium (2:1, v/v), prepared by collecting the conditioned media from Sertoli and germ co-cultures of 10-, 15-, 20-, and 26-day-old rat testes decapsulated and processed as described by Mather and Philips, inMethods in Molecular and Cell Biology, Barnes et al., eds., Alan R. Liss, Inc. (New York, 1984), pp. 29-45, followed by 48 hours of culture in the primary testes cell medium described above.

Binding was performed using 1×10[0329]⁵of the above-described rat germ cells suspended in binding medium [Dulbecco's Modified Eagle's/Ham's F12 nutrient mixture (1:1 v/v) (Gibco) supplemented with 1.2 g/l sodium bicarbonate, 10 mM HEPES buffer, 20 mg/l gentamycin sulfate, and 1 mg/ml bovine serum albumin] with FITC-labeled inhibin A, FITC-labeled activin A, or FITC-labeled activin B (as in Mason, U.S. Pat. No. 4,798,885, supra). Following incubation at 4° C. for 30 minutes the cells plus bound ligand were washed twice with PBS-BSA. Non-specific binding was determined in the presence of 1000-fold excess unconjugated ligand. Binding is reported as fluorescent intensity per cell population minus background fluorescence divided by the percent number of cells per population and corrected for percent competition. Within each experiment duplicate or triplicate analysis of binding was performed. Many independent binding experiments were completed.

FIG. 3 shows the percent competition based on relative fluorescence generated by co-incubations with 1000-fold excess of non-labeled hormone. Surface binding by inhibin A-FITC was competed by unlabeled inhibin A, but not by activin A or activin B. Activin B demonstrated a similar pattern of complete competition in the presence of unlabeled activin B. Activin A was partially competed by activin B and activin A when analyzed for the total germ cell population. Inhibin A did not bind to either of the activin receptors. Inhibin B and activin AB were not tested; it is possible that independent receptors for these species also exist. TGF-β did not compete with inhibin or activin binding. When binding was assessed for individual germ cell stages, similar binding by inhibin A and activin A was found with one exception: activin A did not bind leptotene/zygotene primary spermatocytes.[0330]

I. Conclusions[0331]

There are two separate receptors for activin(s) and inhibin(s) based on the following experimental data obtained:[0332]

1) No cross-competition in binding of FITC-activin A by inhibin A and vice-versa in rat germ cell assay.[0333]

2) Immobilized inhibin A binds a protein that is competed with free inhibin A added to the OβG soluble pituitary membrane extract.[0334]

3) Immobilized activin A binds a protein that is competed by activin A but not inhibin A.[0335]

4) Early pachytene spermatocytes bind inhibin but not activin. Most cells bind both.[0336]

The activin and inhibin receptors are related, as indicated by the fact that they have the same N-terminal amino acid sequence (ValLeuThrGluGluThrGluIleIleMetProThrProLysProGluLeuXaaAlaXaaXaaAsn, which is Sequence Identity No. 1) and they are similar in size (135-150-Kd single band on reduced 10% SDS-PAGE).[0337]

Summarizing the data for the competition studies is the following table:[0338]



Immobilized Ligand	Hormone Spike-Back	Receptor Band

Inhibin A	None	+*
Inhibin A	Inhibin A	−**
Inhibin A	Activin A	not done
Activin A	None	+
Activin A	Inhibin A	+
Activin A	Activin A	−

EXAMPLE IIInhibin/Activin Receptor RNA Probes

A consensus N-terminal oligonucleotide was synthesized using standard DNA synthesis techniques to probe RNA slot blots. The sequence of this oligomer, which is based on the N-terminal sequence of the inhibin/activin receptor and uses mammalian-preferred codons, is as follows:[0339]

5′-TAATTCAGGTTTAGGAGTAGGAGTAGGCATAATAATTTCAGTTTCTTCAGTTAAAAC-3′ (Sequence Identity No. 5)[0340]

An analysis of mRNA expression of the receptor sequence was done using Northern analysis. RNA was prepared from the tissues following homogenization in guanidine isothiocyanate, as described by Chomczynski and Sacchi,[0341]Anal. Biochem.,162: 156-159 (1987). Poly(A)+ RNA was prepared from all sources by chromatography on an oligo(dT)-cellulose column. RNAs were electrophoresed on denaturing formaldehyde-agarose gels and the gels were stained with acridine orange to check for equivalent loading of RNA samples. RNA was transferred to nitrocellulose and hybridized using standard conditions for kinasing oligomers. Slot blots were made by dotting the various RNAs directly onto nitrocellulose and hybridizing directly. These methods are more fully described in Woodruff et al.,Molecular Endocrinol.,1: 561-568 (1987) and Woodruff et al.,Endocrinol.,128: 1647-1654 (1991).

The N-terminal consensus oligomer hybridized most intensely to bovine pituitary RNA and S6BC RNA (a mouse melanoma-derived cell line). It did not hybridize to TM4+/− cAMP mRNA (mouse sertoli-derived cell line) or RL65 mRNA (mouse lung-cell derived cell line), as is clear from FIG. 4.[0342]

By Northern analysis, the mRNA species corresponding to the oligonucleotide is about 6.8 kb in size.[0343]

1 Val Leu Thr Glu Glu Thr Glu Ile Ile Met Pro Thr Pro Lys Pro 1 5 10 15 Glu Leu Xaa AlaXaa Xaa Asn 20

23 amino acids

Amino Acid

Linear

2 Ala Val Asn Pro Thr Gly Arg Asp Ala Val Glu Pro Val Ala Val 1 5 10 15 Xaa Ala Leu Asp LeuIle Asn Lys 20

6 amino acids

Amino Acid

Linear

3 Met Tyr Ala Pro Glu Tyr 1 5

9 amino acids

Amino Acid

Linear

4 Met Ile Phe Ala Leu Leu Phe Phe Gly 1 5

5 TAATTCAGGT TTAGGAGTAGGAGTAGGCAT AATAATTTCA GTTTCTTCAG 50 TTAAAAC 57