CA1305829C - Ovine inhibin - Google Patents
Ovine inhibinInfo
- Publication number
- CA1305829C CA1305829C CA 540422 CA540422A CA1305829C CA 1305829 C CA1305829 C CA 1305829C CA 540422 CA540422 CA 540422 CA 540422 A CA540422 A CA 540422A CA 1305829 C CA1305829 C CA 1305829C
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- CA
- Canada
- Prior art keywords
- inhibin
- protein
- buffer
- chain
- pro
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
OVINE INHIBIN
ABSTRACT OF THE DISCLOSURE
A 34,500-dalton protein with inhibin activity is isolated from ram rete testis fluid using reverse-phase high-performance liquid chromatography and gel filtration. The isolated molecule is composed of two chains having molecular weights of about 18,000 and about 16,500 Daltons, which are bound together by disulfide bonding and the longer of which is likely glycosylated. Microsequencing revealed the NH2-terminal portion of the 18kD chain to be . This 34.5kD
protein specifically inhibits basal secretion of FSH, but not of LH, in a rat anterior pituitary monolayer culture system, exhibiting a half-maximal effective dose of 0.3 ng/ml. Furthermore, antibodies raised against a synthetic replicate of the N-terminal six residues of the 18kD chain are effective to reduce the activity of highly purified 34.5kD ovine inhibin and might be used to exert a profertility effect in rams, ewes and other mammals.
ABSTRACT OF THE DISCLOSURE
A 34,500-dalton protein with inhibin activity is isolated from ram rete testis fluid using reverse-phase high-performance liquid chromatography and gel filtration. The isolated molecule is composed of two chains having molecular weights of about 18,000 and about 16,500 Daltons, which are bound together by disulfide bonding and the longer of which is likely glycosylated. Microsequencing revealed the NH2-terminal portion of the 18kD chain to be . This 34.5kD
protein specifically inhibits basal secretion of FSH, but not of LH, in a rat anterior pituitary monolayer culture system, exhibiting a half-maximal effective dose of 0.3 ng/ml. Furthermore, antibodies raised against a synthetic replicate of the N-terminal six residues of the 18kD chain are effective to reduce the activity of highly purified 34.5kD ovine inhibin and might be used to exert a profertility effect in rams, ewes and other mammals.
Description
OVINE INHIBIN
The present invention relates to a protein having inhibin activity isolated to substantial homogeneity from material obtained from ovine animals.
BACKGROUND O _THE INVENTION
The existence of nhibin as a water-soluble substance of gonadal origin which acts specifically at the pituitary level to suppress the secretion of follicle-stimulating hormone (FSH) was postulated by McCullagh more than 50 years ago, Science, 76, 19-20 (1932). There has been yreat interest in it, and many laboratories have attempted to isolate and characterize this substance. In Nature, 318, 659-663, (1985) Mason et al. published sequences for the subunits of porcine inhibin derived from studies of cDNA. In B.B.R.C., 135, 3, 957-964, March 28, 1986, Mason et al.
published sequences for the subunits of human inhibin similarly derived from cDNA. Inhibin may be used to regulate fertility, gonadotropin secretion or sex hormone production in mammalians, both females and particularly males.
SUMMARY OF THE INVENTION
In accordance with the present invention, a protein having a molecular weight of about 34,500 daltons (34.5kD) and having inhibin activity has been successfully isolated from ram rete testis fluid(RTF).
The protein has been partially characterized using microsequencing methods.
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In a first embodiment, the invention provides a substantially pure ovine inhibin protein consisting essentially of greater than 90~ by weight of total protein, said inhibin protein consisting of a dimer of a first polypeptide chain having an apparent molecular weight of about 18,000 Daltons, as measured by gel electrophoresis, and a second polypeptide chain having a molecular weight of about 16,500 Daltons, as measured by gel electrophoresis, said first chain being linked to said second chain through disulfide bonding, said dimer, when subjected to reverse phase HPLC
elutes from a C8 column of 5 micron particle size and 300 angstrom pore size at between about 27 and about 28 minutes following the instigation of a straight line gradient flow of Buffers A and B, which gradient varies from 25% Buffer B to 90~ Buffer B over a period of 45 minutes, at a flow rate of 0.7 ml per minute and a back pressure of about ~90 psi, with Buffer A being 0.1~ trifluoroacetic acid (TFA) and Buffer B
being 80% acetonitrile in 0.1% TFA;
said first chain having an amino-terminal sequence beginning with Ser-Thr-Pro-Pro-Leu-Pro-Trp-Pro-Trp-Ser-Pro-Ala-Ala-Leu-Arg-Leu-Leu-Gln-Arg-Pro-Pro-Glu-Glu-Pro-Ala-Ala-His-Ala-Asp-Cys-His-Arg-Ala and said second chain having about the formula:
H-Gly-Leu-Glu-Cys-Asp-Gly-Lys-Val-Asn-Ile-Cys-Cys-Lys-Lys-Gln-Phe-Tyr-Val-Ser-Phe-Lys-Asp-Ile-Gly-Trp-Asn-Asp-Trp-Ile-Ile-Ala-Pro-Ser-Gly-Tyr-His-Ala-Asn-Tyr-Cys-Glu-Gly-Glu-Cys-Pro-Ser-His-Ile-Ala-Gly-Thr-Ser-Gly-Ser-Ser-Leu-Ser-Phe-His-Ser-Thr-Val-Ile-Asn-His-Tyr-Arg-Met-Arg-Gly-His-Ser-Pro-Phe-Ala-Asn-Leu-Lys-Ser-Cys-Cys-Val-Pro-Thr-Lys-Leu-Arg-Pro-Met-Ser-Met-Leu-Tyr-Tyr-Asp-Asp-Gly-Gln-Asn-Ile-Ile-Lys-Lys-Asp-Ile-Gln-Asn-Met-Ile-Val-Glu-Glu-Cys-Gly-Cys-Ser-OH, said protein specifically inhibiting basal secretion of follicle-stimulating hormone while not inhibiting basal secretion of luteinizing hormone.
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2a In a second embodiment, the invention provides a protein subunit of ovine inhibin having a purity of greater than 90% by weight based upon total protein selected from the group consisting of (a) a first polypeptide having an apparent molecular weight of about 18kD, as measured by gel electrophoresis, having about 135 amino acid residues, and having an amino terminal sequence beginning with Ser-Thr-Pro-Pro-Leu-Pro-Trp-Pro-Trp-Ser-Pro-Ala-Ala-Leu-Arg-Leu-Leu-Gln-Arg-Pro-Pro-Glu-Glu-Pro-Ala-Ala-His-Ala-Asp-Cys-His-Arg-Ala, and (b) a second polypeptide having an apparent molecular weight of about 16.5kD, as measured by gel electrophoresis, and having the sequence Gly-Leu-Glu-Cys-Asp-Gly-Lys-Val-Asn-Ile-Cys-Cys-Lys-Lys-Gln-Phe-Tyr-Val-Ser-Phe-Lys-Asp-Ile-Gly-Trp-Asn-Asp-Trp-Ile-Ile-Ala~Pro-Ser-Gly-Tyr-His-Ala-Asn-Tyr-Cys-Glu-Gly-Glu-Cys-Pro-Ser-His-Ile-Ala-Gly-Thr-Ser-Gly-Ser-Ser-Leu-Ser-Phe-His-Ser-Thr-Val-Ile-Asn-His-Tyr-Arg-Met-Arg-Gly-His-Ser-Pro-Phe-Ala-Asn-Leu-Lys-Ser-Cys-Cys-Val-Pro-Thr-Lys-Leu-Arg-Pro-Met-Ser-Met-Leu Tyr-Tyr-Asp-Asp-Gly-Gln-Asn-Ile-Ile-Lys-Lys-Asp-Ile-Gln-Asn-Met-Ile-Val-Glu-Glu-Cys-Gly-Cys-Ser, wherein the first polypeptide, when combined through disulfide bonding with the second polypeptide, creates a dimer, which dimer elutes from a Ca reverse phase ~PLC column of 5 micron particle size and 300 angstrom pore size, at between about 27 and about 28 minutes following the instigation of a straight line gradient flow of Buffers A and B, which gradient varies from 25% Buffer B to 90% Buffer B over a period of 45 minutes, at a flow rate of 0.7 ml per minute and a back pressure of about 890 psi, with Buffer A being 0.1% trifluoroacetic acid (TFA) and with Buffer B being 80% acetonitrile in 0.1% TFA.
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Purification of ovine inhibin to substantial homogeneity, i.e., about 90% by weight of total protein in the fraction, was achieved through a combination of protein separation procedures including gel filtration and reverse-phase, high-performance liquid chromatography (RP-HPLC).
BRIEF DESCRIPTION OF DRAWING
FIGURE 1 is a chromatogram of the final RP-HPLC
purification of an inhibin protein active fractions which was applied directly onto a 0.46 x 25 cm Vydac* C8 column with a 5 ~m particle size and a 300A pore size, and eluted at 40~C with a gradient of TFA/CH3CN buffers from 25% Buffer B to 95% Buffer B in 45 minutes, at a flow rate of 0.7 ml/min. with a back pressure of about 890 psi.
DET~ILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Using a multi-step procedure, the 34.5kD peptide was isolated to substantial homogeneity from ram rete testis fluid (RTF). The protein is composed of two chains of 18kD and 16.5kD, and the chains of the intact molecule are held together by disulfide bonding, the linkage between the chains being necessary for biological activity. Amino acid analysis of the total protein has been performed, and a partial amino acid residue sequence of each chain has also been determined, beginning at the amino-terminus. The chains are rich in Cys residues, and it is believed that internal disulfide bonding is also present. The amino-terminal sequence of * trade mark .5~6~
the 18kD chain is Ser-Thr-Pro-Pro-Leu-Pro-Trp-Pro-Trp-Ser-Pro-Ala-Ala-Leu-Arg-Leu-Leu-Gln-Arg-Pro-Pro-Glu-Glu-Pro-Ala-Ala-His~Ala-Asp-Cys. The 18kD chain is estimated to be about 135 residues in length, is likely glycosolated and is linked by one or more disulfide bridges to the 16.5kD chain. The 16.5kD chain has between about 115 and about 130 residues and begins at the N-terminus with the following sequence: Gly-Leu-Glu-Cys-Asp-Gly-Lys-Val-Asn-Ile-Cys-Cys-Lys-Lys-Gln-Phe-(Tyr or Phe). The next residues following these are believed to be: Val-Ser-Phe-Lys-Asp-Ile-Gly. The C-terminus of either chain may be amidated or free acid.
The sharp elution peak of ~he protein which was obtained in the final chromatographic purification step is evidence that the protein has been purified to at least about 90% by weight of total protein. The 34.5kD
protein is water-soluble, and one of the subunits of the native protein is likely glycosylated. A second isolated molecule appears to have an N-shortened version of the 18kD chain, that is shorter by 15 residues, but is linked to an identical 16.5kD chain.
The 34.5kD protein exhibits inhibin activity in that it specifically inhibits basal secretion of FSH but not LH in a rat anterior pituitary monolayer culture system and exhibits a half-maximal effective dose (EC50) of about 0.3 ng/ml ~10 pM.), based upon the assay described in detail in Endocrinology, 113, 1121-31 (1983). The isolated 34.5kD protein, as well as partially purified inhibin preparations, blocks the secretion of both LH and FSH in vitro when cells are stimulated by gonadotropin releasing hormone. In vivo, partially purified inhibin preparations are highly selective to decrease plasma FSH and not LH levels. The effects of inhibin on basal gonadotropin secretion in vitro appears to best reflect the in vivo situation.
The 34.5kD protein is useful for regulating gonadotropin secretion and thus fertility and/or sex hormone 13~)5~
production of both male and female mammalians. The possibility that inhibin might have direct gonadal actions on gametogenesis or steroidogenesis is also likely, and some brain actions of inhibin are suggested.
A purification procedure was used to isolate ovine inhibin from cruds RTF which utilized successive purification steps that include Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) with different stationary phases and/or mobile phases and also include gel filtration or permeation Fast Protein Liquid Chromatography(FPLC).
The starting material for the procedure was about 3950 ml. of RTF that was first dialyzed against Milli Q H20 with 0.02% dimethylsulfide at 4C using dialysis bags with molecular weight cut off (MWC0) ca.
1000. The retentate was divided into two equal batches and lyophilized. Each half of the lyophilized pool was resuspended in column eluant`and subjected to large scale gel permeation using a 5.0 by 150 cm glass column packed with 140 cm Sepharose* CL-6B, Vt=2750 mls.
Column eluant was 6 M ~uanidine HCl, 0.1 M ammonium acetate, 0.05% dimethylsulfide in Milli Q H20, ph 4.75. Æluant was 0.22 ~m filtered and degassed before use. Flow rate was 50 mls/hour. Active fractions from the two batches were pooled and further processed.
Purification by semi-preparative RP-HPLC was carried out first at 50~C. and thereafter at room temperature (RT) using 133 ml-equivalents per run. The inhibin protein fractions from the various columns used for each step are pooled for each following step. Each run was applied directly onto a 1 x 30 cm Vydac* 5-~m particle-si2e C4 column (The Separations Group, Hesperia, CA.) and eluted using a gradient of TEAP
buffer. In this TEAP system, Buffer A consists of 0.1 N.
triethylammonium phosphate(TEAP) pH 5, and Buffer B is 60% CH3CN in Buffer A. After all the filtrate had been loaded, the column was washed with the aqueous * trade mark 1~05~
the column is maintained at 2.5 ml per minute. The column is loaded at 30% Buffer B, and a gradient for the mobile phase was then begun gradually changing to 95%
over 30 minutes. The fractions are separated by an Altex* 420 gradient liquid chromatography system equipped with a Spectroflow* 773 W detector (Kratos Analytical Instruments, Ramsey, N. J.) and a Servocoder SR 6253 strip chart recorder and are collected and tested for substantial inhibin activity.
Inhibin protein fractions from the various individual columns were pooled and further purified by a 1 x 30 cm Vydac* 5-~m-particle-size C4 column and a heptafluorobutyric acid (HFBA) buffer system at RT. In the HFBA system, Buffer A contains 1 ml of HFBA in 999 ml water, and Buffer B is 400 ml of water, 1 ml of HFBA and 599 ml of acetonitrile. Columns were loaded at 30% B
followed by a gradient to 58% B in 25 minutes.
Active zones from reversed phase HPLC were lyophilized and resuspended in column eluant for processing on Pharmacia FPLC system by applying to two 1 x 30 cm Superose*(FPLC) 12-B columns, 10 ~m (Pharmacia Fine Chemicals, Piscataway, N. J.) linked in series.
Each column was eluted with 6M guanidine HCl, O.lM
ammonium acetate, pH 4.75, and 0.5~ DMS in Milli ~ H20 at a flow rate of 0.4 ml per minute for about 50 min.
The column fractions were monitored by UV absorption and by bioassay. Active fractions eluted between KAV=0.31-0.36.
Inhibin protein fractions from the various individual columns were pooled and further purified by RP-HPLC using 5-~m-particle-size C4 column, 1 x 30 cm, and 0.5% TFA/CH3CN buffer system.
After such ~PLC desalting, the active fraction was lyophilized and subjected to FPLC cation exchange by being brought up in Buffer A which was 50 mM sodium acetate, 4 M urea, ~ mM CHAPS (3-[(Cholamidopropyl) dimethylammonio]-l-propanesulfonate) in Milli Q H20, * trade mark pH 5.3. Buffer B was 1 M NaCl in Buffer A. A Pharmacia FPLC system equipped with a Mono S HR 5/5 column, Vt = 1 ml, was used at a flow rate of l ml per min.
Column was loaded at 0% B followed by a gradient to 30% B
in 25 minutes and then to lOQ% B in 5 minutes.
Next, the active fraction was applied to a 0.46 x 25 cm Vydac* C8 Column of reversed phase material with a 5 ~m particle SiZ8 and a 300A pore size.
Buffer A is 0.5% (v/v) TFA in water and Buffer B is 1 ml TFA, 200 ml of water and 799 ml of acetonitrile.
Flow rate was 0.7 ml/min at 40C. with a back pressure of 900 psi. Buffer B was used at 25 volume % for the initial loading, followed by a gradient to 50% in 25 minutes. Two zones of active inhibin protein eluted, and both were separately processed thereafter.
Each active fraction was applied to a 0.46 x 25 cm Vydac* C8 Column of reversed phase material with a 5 ~m particle size and a 300A pore size.
Buffer A is 0.1% (v/v) TFA in water, and Buffer B is 1 ml TFA, 200 ml of water and 799 ml of acetonitrile.
Buffer B was used at 25 volume % for the initial loading at 40C and a flow rate of 1.2 ml/min. Then a gradient to 95% is run in 45 minutes at a flow rate of 0.7 ml/min at 40C with a back pressure of 890 psi, and a detector setting of 215 nm, 2.9 AUFS, which was slightly ch~nged to 214 nm. before elution of the peak. The purified inhibin protein eluted generally between about 27.0 minutes and about 28.3 minutes after start of the gradient, which is equal to between about 18.9 ml and about 19.8 ml of elutant after start of the gradient. A
chromatogram of the final step, for the later eluting active fraction from the previous step, is depicted in FIGURE 1 and was generated using an Altex* 420 System, two Beckman* Model lOOA pumps, a Datamark* Servocoder SR
6253 strip chart recorder, a Kratos Spectroflow* 773 variable wavelength, W/visible detector and a Rheodyne 7125 injector with a 2.0 ml loop.
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The inhibin protein fractions accumulated from all batches, respectively for the earlier and later eluting fractions, were pooled, resulting in a total of approximately 195 pg of inhibin protein, including about 120 ~g of the earlier eluting active inhibin fraction and about 75 ~g of the later eluting fraction. The 195 ~ug was estimated to have been obtained from about 2700 ml of RTF, with the remainder having been used in the biological testing that was carried out to identify the active fractions during purification.
Amino acid analyses of the substantially homogeneous, later eluting inhibin protein was performed after acid hydrolysis in 4M methane sulfonic acid and 0.2% tryptamine at 110C. for 24 hours. Norleucine was used as an internal standard. The amino acid analyzer was a Beckman Model 121M with ninhydrin post-column derivatization. The results are shown in the Table below.
TABLE
AMINO ACID COMPOSITION OF PURIFIED INHIBIN
PROTEIN FROM RAM RETE TESTIS FLUID
Amino AcidInhibin Protein Asx 22 Thr 11 Ser 17 Glx 22 Gly 16 Ala 16 ~al 11 Met 5 Ile 8 Leu 22 Tyr 8 Phe 10 His 8 Trp 4 ~ ;~O~S8~:~9 Amino Acid Inhibin Protein Lys 10 Arg 10 Cys 12 Pro 23 Although the results of amino acid analysis of a large protein are by no means definitive of the precise composition because amino acid analysis can only provide a rough estimate, the results give a fairly accurate portrayal of relative residue compositions and together with other SDS and sequence data accura~ely define a pure compound.
A portion of the purified, later eluting fraction highest peak was exposed to 2% sodium dodecyl sulfate (SDS) with and without 5% ~-mercaptoethanol at neutral p~ in a boiling water bath for 2-3 minutes; both aliquots were subsequently applied to a slab gel and subjected to SDS polyacrylamide gel electrophoresis (PAGE) as described by Laemmli, U.K.I Nature, 227, 680-685(1970). Protein bands were discovered by silver staining.
On SDS-PAGE under non-reducing condition, the inhibin protein showed a single band migratin~ at about 34-5kD. Under reducing condition, the inhibin protein separated into two bands, one migrating at 18kD and the other at 16.5kD. Electrophoresis showed the protein was more than 90% pure.
NH2-terminal sequence analyses of the 18kD
and 16.5kD chains of the 34.5kD inhibin protein were accomplished by first separating the two chains by SDS-PAGE under reducing conditions. Microsequencing, as described in Spiess, J. et al. Biochemistry, 20, 1982-1988 (1981), of the intact inhibin protein beginning at the NH2-terminus consistently revealed two residues of approximately equal concentration at every cycle, indicating that the protein is composed of 58~3 g two chains. Based upon the results from multiple sequencing analyses of both the intact and reduced inhibin protein, the sequence of the NH2-terminal residues of the 18kD chain of the inhibin protein is Ser-Thr-Pro-Pro-Leu-Pro-Trp-Pro-Trp-Ser-Pro-Ala-Ala-Leu-Arg-Leu-Leu-Gln-Arg-Pro-Pro-Glu-Glu-Pro-Ala-Ala-His-Ala-Asp-Cys. The first NH2-terminal residues of the 16.5kD chain of the inhibin protein are Gly-Leu-Glu-Cys, and it is believed that the next residues are Asp-Gly-Lys-Val-Asn-Ile-Cys-Cys-Lys-Lys-Gln-Phe-(Tyr or Phe). The next residues following these are believed to be: Val-Ser-Phe-Lys-Asp-Ile-Gly~ The C-terminal portion of the 16.5KD chain is believed to be: Trp-Asn-Asp-Trp-Ile-Ile-Ala-Pro-Ser-Gly-Tyr-His-Ala-Asn-Tyr-Cys-Glu-Gly-Glu-Cys-Pro-Ser-His-Ile-Ala-Gly-Thr-Ser-Gly-Ser-Ser-Leu-Ser-Phe-His-Ser-Thr-Val-Ile-Asn-His-Tyr-Arg-Met-Arg-Gly-His-Ser-Pro-Phe-Ala-Asn-Leu-Lys-Ser-Cys-Cys-Val-Pro-Thr-Lys-Leu-Arg-Pro-Met-Ser-Met-Leu-Tyr-Tyr-Asp-Asp-Gly-Gln-Asn-Ile-Ile-Lys-Lys-Asp-Ile-Gln-Asn-Met-Ile-Val-Glu-Glu-Cys-Gly-Cys-Ser-OH.
Because a substantial portion of the sequence of both chains of an inhibin protein is accurately known, the mRNA encoding the chains can be isolated, and the cDNA's can be synthesized by recombinant DNA
techniques. Messenger RNA (mRNA) is obtained from ovarian follicules or from ram testes which produce inhibin, and then cDNA is synthesized from the mRNA by reverse transcription. The cDNA is inserted into a cloning vector which is used to transform a suitable host to create a cDNA library.
Based upon the known partial amino acid residue sequence of the inhibin chains, labelled oligonucleotides are synthesized for detecting cDNA corresponding to each chain. Because of the degeneracy of the genetic code, mixed hybridization probes are prepared and used as probes. These probes are then used to select, from the library, cDNA clones that contain gene sequences encoding the chains. cDNA libraries may also be screened by :1 ~05~
immunological expression assay with an antibody raised against inhibin or one of the two inhibin chains.
Immunological expression assay may also be used to confirm screening ~ith hybridization probes.
From selected clones, cDNA is excised and inserted into appropriate vectors under the control of suitable promotor sequences, and the vectors are transformed into cell lines for expression of the recombinant inhibin chains. Although vectors containing the genes for both chains could conceivably be transformed into the same cell line, for simplicity, vectors for expression of each chain are preferably transformed separately into cell lines. The two inhibin chains can then be isolated from the cellular material and/or the cell culture medium. The two chains are then subjected to oxidizing conditions which promote disulfide bonding between the chains. The foregoing molecular biology techniques may also be used to read the gene sequences encoding the separate inhibin chains, and thereby completely characterize the protein chains.
Substantially pure 34.5kD inhibin or the nontoxic salts thereof, combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition, may be administered to mammals, including humans, either intravenously, subcutaneously, percutaneously, intramuscularly or orally for control of fertility, gonadotropin secretion or sex hormone production.
Furthermore antibodies raised against synthetic fragments of inhibin, e.g. the six N-terminal residues of the 18KD chain, na~e~y Se~-Thr-Pro-Pro-Leu-Pro, have been shown to neutralize the activity of purified inhibin. Thus passive (administration of antibodies) or active (administration of immunogenic inhibin as antigen) immunization methods could be employed to block endogenous inhibin and thereby elevate endogenous gonadotropin secretion and exert a profertility effect in sheep (both rams and ewes~, in human beings and in i :~C~5&2~
other vertibrate animal species having inhibin of a similar polypeptide structure. Administration of inhibin ind~ces decreased fertility in female mammals and decreases spermatogenesis in male mammals, and administration of a sufficient amount of inhibin could be employed to induce infertility in sheep, including rams and ewes, and in other mammals. Inhibin is also useful for tests to diagnose infertility.
Such peptides are often administered in the form of pharmaceutically acceptable nontoxic salts, such as acid addition salts or metal complexes, e.g., with zinc, iron or the like (which are considered as salts for purposes of this application). Illustrative of such acid addition salts are hydrochloride, hydrobromide, sulphate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate and the like. If administration in liquid form is desired, sweetening and/or flavoring may be used, and intravenous administration in isotonic saline, phosphate buffer solutions or the like may be effected.
Inhibin should be administered under the guidance of a veterinarian or a physician, and pharmaceutical compositions will usually contain an effective amount of the peptide in conjunction with a conventional, pharmaceutically-acceptable carrier. The dosage will vary depending upon the specific purpose for which the protein is being administered, and dosage levels in the range of about 0.1 to about 1 milligrams per Kg. of body weight may be used when the protein is administered on a regular basis as a male contraceptive.
Although the method of purification of inhibin has been described primarily in terms of isolation from RTF, inhibin can be similarly purified from other crude extracts, for example follicular fluid.
Although the invention has been described with regard to its preferred embodiments, which constitute the best mode presently known to the inventors~ it ~0~ 2~
should be understood that various changes and modifications as would be obvious to one having the ordinary skill in this art may be made without departing from the scope of the invention which is set forth in the claims appended hereto.
The present invention relates to a protein having inhibin activity isolated to substantial homogeneity from material obtained from ovine animals.
BACKGROUND O _THE INVENTION
The existence of nhibin as a water-soluble substance of gonadal origin which acts specifically at the pituitary level to suppress the secretion of follicle-stimulating hormone (FSH) was postulated by McCullagh more than 50 years ago, Science, 76, 19-20 (1932). There has been yreat interest in it, and many laboratories have attempted to isolate and characterize this substance. In Nature, 318, 659-663, (1985) Mason et al. published sequences for the subunits of porcine inhibin derived from studies of cDNA. In B.B.R.C., 135, 3, 957-964, March 28, 1986, Mason et al.
published sequences for the subunits of human inhibin similarly derived from cDNA. Inhibin may be used to regulate fertility, gonadotropin secretion or sex hormone production in mammalians, both females and particularly males.
SUMMARY OF THE INVENTION
In accordance with the present invention, a protein having a molecular weight of about 34,500 daltons (34.5kD) and having inhibin activity has been successfully isolated from ram rete testis fluid(RTF).
The protein has been partially characterized using microsequencing methods.
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In a first embodiment, the invention provides a substantially pure ovine inhibin protein consisting essentially of greater than 90~ by weight of total protein, said inhibin protein consisting of a dimer of a first polypeptide chain having an apparent molecular weight of about 18,000 Daltons, as measured by gel electrophoresis, and a second polypeptide chain having a molecular weight of about 16,500 Daltons, as measured by gel electrophoresis, said first chain being linked to said second chain through disulfide bonding, said dimer, when subjected to reverse phase HPLC
elutes from a C8 column of 5 micron particle size and 300 angstrom pore size at between about 27 and about 28 minutes following the instigation of a straight line gradient flow of Buffers A and B, which gradient varies from 25% Buffer B to 90~ Buffer B over a period of 45 minutes, at a flow rate of 0.7 ml per minute and a back pressure of about ~90 psi, with Buffer A being 0.1~ trifluoroacetic acid (TFA) and Buffer B
being 80% acetonitrile in 0.1% TFA;
said first chain having an amino-terminal sequence beginning with Ser-Thr-Pro-Pro-Leu-Pro-Trp-Pro-Trp-Ser-Pro-Ala-Ala-Leu-Arg-Leu-Leu-Gln-Arg-Pro-Pro-Glu-Glu-Pro-Ala-Ala-His-Ala-Asp-Cys-His-Arg-Ala and said second chain having about the formula:
H-Gly-Leu-Glu-Cys-Asp-Gly-Lys-Val-Asn-Ile-Cys-Cys-Lys-Lys-Gln-Phe-Tyr-Val-Ser-Phe-Lys-Asp-Ile-Gly-Trp-Asn-Asp-Trp-Ile-Ile-Ala-Pro-Ser-Gly-Tyr-His-Ala-Asn-Tyr-Cys-Glu-Gly-Glu-Cys-Pro-Ser-His-Ile-Ala-Gly-Thr-Ser-Gly-Ser-Ser-Leu-Ser-Phe-His-Ser-Thr-Val-Ile-Asn-His-Tyr-Arg-Met-Arg-Gly-His-Ser-Pro-Phe-Ala-Asn-Leu-Lys-Ser-Cys-Cys-Val-Pro-Thr-Lys-Leu-Arg-Pro-Met-Ser-Met-Leu-Tyr-Tyr-Asp-Asp-Gly-Gln-Asn-Ile-Ile-Lys-Lys-Asp-Ile-Gln-Asn-Met-Ile-Val-Glu-Glu-Cys-Gly-Cys-Ser-OH, said protein specifically inhibiting basal secretion of follicle-stimulating hormone while not inhibiting basal secretion of luteinizing hormone.
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2a In a second embodiment, the invention provides a protein subunit of ovine inhibin having a purity of greater than 90% by weight based upon total protein selected from the group consisting of (a) a first polypeptide having an apparent molecular weight of about 18kD, as measured by gel electrophoresis, having about 135 amino acid residues, and having an amino terminal sequence beginning with Ser-Thr-Pro-Pro-Leu-Pro-Trp-Pro-Trp-Ser-Pro-Ala-Ala-Leu-Arg-Leu-Leu-Gln-Arg-Pro-Pro-Glu-Glu-Pro-Ala-Ala-His-Ala-Asp-Cys-His-Arg-Ala, and (b) a second polypeptide having an apparent molecular weight of about 16.5kD, as measured by gel electrophoresis, and having the sequence Gly-Leu-Glu-Cys-Asp-Gly-Lys-Val-Asn-Ile-Cys-Cys-Lys-Lys-Gln-Phe-Tyr-Val-Ser-Phe-Lys-Asp-Ile-Gly-Trp-Asn-Asp-Trp-Ile-Ile-Ala~Pro-Ser-Gly-Tyr-His-Ala-Asn-Tyr-Cys-Glu-Gly-Glu-Cys-Pro-Ser-His-Ile-Ala-Gly-Thr-Ser-Gly-Ser-Ser-Leu-Ser-Phe-His-Ser-Thr-Val-Ile-Asn-His-Tyr-Arg-Met-Arg-Gly-His-Ser-Pro-Phe-Ala-Asn-Leu-Lys-Ser-Cys-Cys-Val-Pro-Thr-Lys-Leu-Arg-Pro-Met-Ser-Met-Leu Tyr-Tyr-Asp-Asp-Gly-Gln-Asn-Ile-Ile-Lys-Lys-Asp-Ile-Gln-Asn-Met-Ile-Val-Glu-Glu-Cys-Gly-Cys-Ser, wherein the first polypeptide, when combined through disulfide bonding with the second polypeptide, creates a dimer, which dimer elutes from a Ca reverse phase ~PLC column of 5 micron particle size and 300 angstrom pore size, at between about 27 and about 28 minutes following the instigation of a straight line gradient flow of Buffers A and B, which gradient varies from 25% Buffer B to 90% Buffer B over a period of 45 minutes, at a flow rate of 0.7 ml per minute and a back pressure of about 890 psi, with Buffer A being 0.1% trifluoroacetic acid (TFA) and with Buffer B being 80% acetonitrile in 0.1% TFA.
JJ:
~~
130~9 2~
Purification of ovine inhibin to substantial homogeneity, i.e., about 90% by weight of total protein in the fraction, was achieved through a combination of protein separation procedures including gel filtration and reverse-phase, high-performance liquid chromatography (RP-HPLC).
BRIEF DESCRIPTION OF DRAWING
FIGURE 1 is a chromatogram of the final RP-HPLC
purification of an inhibin protein active fractions which was applied directly onto a 0.46 x 25 cm Vydac* C8 column with a 5 ~m particle size and a 300A pore size, and eluted at 40~C with a gradient of TFA/CH3CN buffers from 25% Buffer B to 95% Buffer B in 45 minutes, at a flow rate of 0.7 ml/min. with a back pressure of about 890 psi.
DET~ILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Using a multi-step procedure, the 34.5kD peptide was isolated to substantial homogeneity from ram rete testis fluid (RTF). The protein is composed of two chains of 18kD and 16.5kD, and the chains of the intact molecule are held together by disulfide bonding, the linkage between the chains being necessary for biological activity. Amino acid analysis of the total protein has been performed, and a partial amino acid residue sequence of each chain has also been determined, beginning at the amino-terminus. The chains are rich in Cys residues, and it is believed that internal disulfide bonding is also present. The amino-terminal sequence of * trade mark .5~6~
the 18kD chain is Ser-Thr-Pro-Pro-Leu-Pro-Trp-Pro-Trp-Ser-Pro-Ala-Ala-Leu-Arg-Leu-Leu-Gln-Arg-Pro-Pro-Glu-Glu-Pro-Ala-Ala-His~Ala-Asp-Cys. The 18kD chain is estimated to be about 135 residues in length, is likely glycosolated and is linked by one or more disulfide bridges to the 16.5kD chain. The 16.5kD chain has between about 115 and about 130 residues and begins at the N-terminus with the following sequence: Gly-Leu-Glu-Cys-Asp-Gly-Lys-Val-Asn-Ile-Cys-Cys-Lys-Lys-Gln-Phe-(Tyr or Phe). The next residues following these are believed to be: Val-Ser-Phe-Lys-Asp-Ile-Gly. The C-terminus of either chain may be amidated or free acid.
The sharp elution peak of ~he protein which was obtained in the final chromatographic purification step is evidence that the protein has been purified to at least about 90% by weight of total protein. The 34.5kD
protein is water-soluble, and one of the subunits of the native protein is likely glycosylated. A second isolated molecule appears to have an N-shortened version of the 18kD chain, that is shorter by 15 residues, but is linked to an identical 16.5kD chain.
The 34.5kD protein exhibits inhibin activity in that it specifically inhibits basal secretion of FSH but not LH in a rat anterior pituitary monolayer culture system and exhibits a half-maximal effective dose (EC50) of about 0.3 ng/ml ~10 pM.), based upon the assay described in detail in Endocrinology, 113, 1121-31 (1983). The isolated 34.5kD protein, as well as partially purified inhibin preparations, blocks the secretion of both LH and FSH in vitro when cells are stimulated by gonadotropin releasing hormone. In vivo, partially purified inhibin preparations are highly selective to decrease plasma FSH and not LH levels. The effects of inhibin on basal gonadotropin secretion in vitro appears to best reflect the in vivo situation.
The 34.5kD protein is useful for regulating gonadotropin secretion and thus fertility and/or sex hormone 13~)5~
production of both male and female mammalians. The possibility that inhibin might have direct gonadal actions on gametogenesis or steroidogenesis is also likely, and some brain actions of inhibin are suggested.
A purification procedure was used to isolate ovine inhibin from cruds RTF which utilized successive purification steps that include Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) with different stationary phases and/or mobile phases and also include gel filtration or permeation Fast Protein Liquid Chromatography(FPLC).
The starting material for the procedure was about 3950 ml. of RTF that was first dialyzed against Milli Q H20 with 0.02% dimethylsulfide at 4C using dialysis bags with molecular weight cut off (MWC0) ca.
1000. The retentate was divided into two equal batches and lyophilized. Each half of the lyophilized pool was resuspended in column eluant`and subjected to large scale gel permeation using a 5.0 by 150 cm glass column packed with 140 cm Sepharose* CL-6B, Vt=2750 mls.
Column eluant was 6 M ~uanidine HCl, 0.1 M ammonium acetate, 0.05% dimethylsulfide in Milli Q H20, ph 4.75. Æluant was 0.22 ~m filtered and degassed before use. Flow rate was 50 mls/hour. Active fractions from the two batches were pooled and further processed.
Purification by semi-preparative RP-HPLC was carried out first at 50~C. and thereafter at room temperature (RT) using 133 ml-equivalents per run. The inhibin protein fractions from the various columns used for each step are pooled for each following step. Each run was applied directly onto a 1 x 30 cm Vydac* 5-~m particle-si2e C4 column (The Separations Group, Hesperia, CA.) and eluted using a gradient of TEAP
buffer. In this TEAP system, Buffer A consists of 0.1 N.
triethylammonium phosphate(TEAP) pH 5, and Buffer B is 60% CH3CN in Buffer A. After all the filtrate had been loaded, the column was washed with the aqueous * trade mark 1~05~
the column is maintained at 2.5 ml per minute. The column is loaded at 30% Buffer B, and a gradient for the mobile phase was then begun gradually changing to 95%
over 30 minutes. The fractions are separated by an Altex* 420 gradient liquid chromatography system equipped with a Spectroflow* 773 W detector (Kratos Analytical Instruments, Ramsey, N. J.) and a Servocoder SR 6253 strip chart recorder and are collected and tested for substantial inhibin activity.
Inhibin protein fractions from the various individual columns were pooled and further purified by a 1 x 30 cm Vydac* 5-~m-particle-size C4 column and a heptafluorobutyric acid (HFBA) buffer system at RT. In the HFBA system, Buffer A contains 1 ml of HFBA in 999 ml water, and Buffer B is 400 ml of water, 1 ml of HFBA and 599 ml of acetonitrile. Columns were loaded at 30% B
followed by a gradient to 58% B in 25 minutes.
Active zones from reversed phase HPLC were lyophilized and resuspended in column eluant for processing on Pharmacia FPLC system by applying to two 1 x 30 cm Superose*(FPLC) 12-B columns, 10 ~m (Pharmacia Fine Chemicals, Piscataway, N. J.) linked in series.
Each column was eluted with 6M guanidine HCl, O.lM
ammonium acetate, pH 4.75, and 0.5~ DMS in Milli ~ H20 at a flow rate of 0.4 ml per minute for about 50 min.
The column fractions were monitored by UV absorption and by bioassay. Active fractions eluted between KAV=0.31-0.36.
Inhibin protein fractions from the various individual columns were pooled and further purified by RP-HPLC using 5-~m-particle-size C4 column, 1 x 30 cm, and 0.5% TFA/CH3CN buffer system.
After such ~PLC desalting, the active fraction was lyophilized and subjected to FPLC cation exchange by being brought up in Buffer A which was 50 mM sodium acetate, 4 M urea, ~ mM CHAPS (3-[(Cholamidopropyl) dimethylammonio]-l-propanesulfonate) in Milli Q H20, * trade mark pH 5.3. Buffer B was 1 M NaCl in Buffer A. A Pharmacia FPLC system equipped with a Mono S HR 5/5 column, Vt = 1 ml, was used at a flow rate of l ml per min.
Column was loaded at 0% B followed by a gradient to 30% B
in 25 minutes and then to lOQ% B in 5 minutes.
Next, the active fraction was applied to a 0.46 x 25 cm Vydac* C8 Column of reversed phase material with a 5 ~m particle SiZ8 and a 300A pore size.
Buffer A is 0.5% (v/v) TFA in water and Buffer B is 1 ml TFA, 200 ml of water and 799 ml of acetonitrile.
Flow rate was 0.7 ml/min at 40C. with a back pressure of 900 psi. Buffer B was used at 25 volume % for the initial loading, followed by a gradient to 50% in 25 minutes. Two zones of active inhibin protein eluted, and both were separately processed thereafter.
Each active fraction was applied to a 0.46 x 25 cm Vydac* C8 Column of reversed phase material with a 5 ~m particle size and a 300A pore size.
Buffer A is 0.1% (v/v) TFA in water, and Buffer B is 1 ml TFA, 200 ml of water and 799 ml of acetonitrile.
Buffer B was used at 25 volume % for the initial loading at 40C and a flow rate of 1.2 ml/min. Then a gradient to 95% is run in 45 minutes at a flow rate of 0.7 ml/min at 40C with a back pressure of 890 psi, and a detector setting of 215 nm, 2.9 AUFS, which was slightly ch~nged to 214 nm. before elution of the peak. The purified inhibin protein eluted generally between about 27.0 minutes and about 28.3 minutes after start of the gradient, which is equal to between about 18.9 ml and about 19.8 ml of elutant after start of the gradient. A
chromatogram of the final step, for the later eluting active fraction from the previous step, is depicted in FIGURE 1 and was generated using an Altex* 420 System, two Beckman* Model lOOA pumps, a Datamark* Servocoder SR
6253 strip chart recorder, a Kratos Spectroflow* 773 variable wavelength, W/visible detector and a Rheodyne 7125 injector with a 2.0 ml loop.
* trade mark ~., 82~
The inhibin protein fractions accumulated from all batches, respectively for the earlier and later eluting fractions, were pooled, resulting in a total of approximately 195 pg of inhibin protein, including about 120 ~g of the earlier eluting active inhibin fraction and about 75 ~g of the later eluting fraction. The 195 ~ug was estimated to have been obtained from about 2700 ml of RTF, with the remainder having been used in the biological testing that was carried out to identify the active fractions during purification.
Amino acid analyses of the substantially homogeneous, later eluting inhibin protein was performed after acid hydrolysis in 4M methane sulfonic acid and 0.2% tryptamine at 110C. for 24 hours. Norleucine was used as an internal standard. The amino acid analyzer was a Beckman Model 121M with ninhydrin post-column derivatization. The results are shown in the Table below.
TABLE
AMINO ACID COMPOSITION OF PURIFIED INHIBIN
PROTEIN FROM RAM RETE TESTIS FLUID
Amino AcidInhibin Protein Asx 22 Thr 11 Ser 17 Glx 22 Gly 16 Ala 16 ~al 11 Met 5 Ile 8 Leu 22 Tyr 8 Phe 10 His 8 Trp 4 ~ ;~O~S8~:~9 Amino Acid Inhibin Protein Lys 10 Arg 10 Cys 12 Pro 23 Although the results of amino acid analysis of a large protein are by no means definitive of the precise composition because amino acid analysis can only provide a rough estimate, the results give a fairly accurate portrayal of relative residue compositions and together with other SDS and sequence data accura~ely define a pure compound.
A portion of the purified, later eluting fraction highest peak was exposed to 2% sodium dodecyl sulfate (SDS) with and without 5% ~-mercaptoethanol at neutral p~ in a boiling water bath for 2-3 minutes; both aliquots were subsequently applied to a slab gel and subjected to SDS polyacrylamide gel electrophoresis (PAGE) as described by Laemmli, U.K.I Nature, 227, 680-685(1970). Protein bands were discovered by silver staining.
On SDS-PAGE under non-reducing condition, the inhibin protein showed a single band migratin~ at about 34-5kD. Under reducing condition, the inhibin protein separated into two bands, one migrating at 18kD and the other at 16.5kD. Electrophoresis showed the protein was more than 90% pure.
NH2-terminal sequence analyses of the 18kD
and 16.5kD chains of the 34.5kD inhibin protein were accomplished by first separating the two chains by SDS-PAGE under reducing conditions. Microsequencing, as described in Spiess, J. et al. Biochemistry, 20, 1982-1988 (1981), of the intact inhibin protein beginning at the NH2-terminus consistently revealed two residues of approximately equal concentration at every cycle, indicating that the protein is composed of 58~3 g two chains. Based upon the results from multiple sequencing analyses of both the intact and reduced inhibin protein, the sequence of the NH2-terminal residues of the 18kD chain of the inhibin protein is Ser-Thr-Pro-Pro-Leu-Pro-Trp-Pro-Trp-Ser-Pro-Ala-Ala-Leu-Arg-Leu-Leu-Gln-Arg-Pro-Pro-Glu-Glu-Pro-Ala-Ala-His-Ala-Asp-Cys. The first NH2-terminal residues of the 16.5kD chain of the inhibin protein are Gly-Leu-Glu-Cys, and it is believed that the next residues are Asp-Gly-Lys-Val-Asn-Ile-Cys-Cys-Lys-Lys-Gln-Phe-(Tyr or Phe). The next residues following these are believed to be: Val-Ser-Phe-Lys-Asp-Ile-Gly~ The C-terminal portion of the 16.5KD chain is believed to be: Trp-Asn-Asp-Trp-Ile-Ile-Ala-Pro-Ser-Gly-Tyr-His-Ala-Asn-Tyr-Cys-Glu-Gly-Glu-Cys-Pro-Ser-His-Ile-Ala-Gly-Thr-Ser-Gly-Ser-Ser-Leu-Ser-Phe-His-Ser-Thr-Val-Ile-Asn-His-Tyr-Arg-Met-Arg-Gly-His-Ser-Pro-Phe-Ala-Asn-Leu-Lys-Ser-Cys-Cys-Val-Pro-Thr-Lys-Leu-Arg-Pro-Met-Ser-Met-Leu-Tyr-Tyr-Asp-Asp-Gly-Gln-Asn-Ile-Ile-Lys-Lys-Asp-Ile-Gln-Asn-Met-Ile-Val-Glu-Glu-Cys-Gly-Cys-Ser-OH.
Because a substantial portion of the sequence of both chains of an inhibin protein is accurately known, the mRNA encoding the chains can be isolated, and the cDNA's can be synthesized by recombinant DNA
techniques. Messenger RNA (mRNA) is obtained from ovarian follicules or from ram testes which produce inhibin, and then cDNA is synthesized from the mRNA by reverse transcription. The cDNA is inserted into a cloning vector which is used to transform a suitable host to create a cDNA library.
Based upon the known partial amino acid residue sequence of the inhibin chains, labelled oligonucleotides are synthesized for detecting cDNA corresponding to each chain. Because of the degeneracy of the genetic code, mixed hybridization probes are prepared and used as probes. These probes are then used to select, from the library, cDNA clones that contain gene sequences encoding the chains. cDNA libraries may also be screened by :1 ~05~
immunological expression assay with an antibody raised against inhibin or one of the two inhibin chains.
Immunological expression assay may also be used to confirm screening ~ith hybridization probes.
From selected clones, cDNA is excised and inserted into appropriate vectors under the control of suitable promotor sequences, and the vectors are transformed into cell lines for expression of the recombinant inhibin chains. Although vectors containing the genes for both chains could conceivably be transformed into the same cell line, for simplicity, vectors for expression of each chain are preferably transformed separately into cell lines. The two inhibin chains can then be isolated from the cellular material and/or the cell culture medium. The two chains are then subjected to oxidizing conditions which promote disulfide bonding between the chains. The foregoing molecular biology techniques may also be used to read the gene sequences encoding the separate inhibin chains, and thereby completely characterize the protein chains.
Substantially pure 34.5kD inhibin or the nontoxic salts thereof, combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition, may be administered to mammals, including humans, either intravenously, subcutaneously, percutaneously, intramuscularly or orally for control of fertility, gonadotropin secretion or sex hormone production.
Furthermore antibodies raised against synthetic fragments of inhibin, e.g. the six N-terminal residues of the 18KD chain, na~e~y Se~-Thr-Pro-Pro-Leu-Pro, have been shown to neutralize the activity of purified inhibin. Thus passive (administration of antibodies) or active (administration of immunogenic inhibin as antigen) immunization methods could be employed to block endogenous inhibin and thereby elevate endogenous gonadotropin secretion and exert a profertility effect in sheep (both rams and ewes~, in human beings and in i :~C~5&2~
other vertibrate animal species having inhibin of a similar polypeptide structure. Administration of inhibin ind~ces decreased fertility in female mammals and decreases spermatogenesis in male mammals, and administration of a sufficient amount of inhibin could be employed to induce infertility in sheep, including rams and ewes, and in other mammals. Inhibin is also useful for tests to diagnose infertility.
Such peptides are often administered in the form of pharmaceutically acceptable nontoxic salts, such as acid addition salts or metal complexes, e.g., with zinc, iron or the like (which are considered as salts for purposes of this application). Illustrative of such acid addition salts are hydrochloride, hydrobromide, sulphate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate and the like. If administration in liquid form is desired, sweetening and/or flavoring may be used, and intravenous administration in isotonic saline, phosphate buffer solutions or the like may be effected.
Inhibin should be administered under the guidance of a veterinarian or a physician, and pharmaceutical compositions will usually contain an effective amount of the peptide in conjunction with a conventional, pharmaceutically-acceptable carrier. The dosage will vary depending upon the specific purpose for which the protein is being administered, and dosage levels in the range of about 0.1 to about 1 milligrams per Kg. of body weight may be used when the protein is administered on a regular basis as a male contraceptive.
Although the method of purification of inhibin has been described primarily in terms of isolation from RTF, inhibin can be similarly purified from other crude extracts, for example follicular fluid.
Although the invention has been described with regard to its preferred embodiments, which constitute the best mode presently known to the inventors~ it ~0~ 2~
should be understood that various changes and modifications as would be obvious to one having the ordinary skill in this art may be made without departing from the scope of the invention which is set forth in the claims appended hereto.
Claims (4)
1. A substantially pure ovine inhibin protein consisting essentially of greater than 90% by weight of total protein, said inhibin protein consisting of a dimer of a first polypeptide chain having an apparent molecular weight of about 18,000 Daltons, as measured by gel electrophoresis, and a second polypeptide chain having a molecular weight of about 16,500 Daltons, as measured by gel electrophoresis, said first chain being linked to said second chain through disulfide bonding, said dimer, when subjected to reverse phase HPLC
elutes from a C8 column of 5 micron particle size and 300 angstrom pore size at between about 27 and about 28 minutes following the instigation of a straight line gradient flow of Buffers A and B, which gradient varies from 25% Buffer B to 90% Buffer B over a period of 45 minutes, at a flow rate of 0.7 ml per minute and a back pressure of about 890 psi, with Buffer A being 0.1% trifluoroacetic acid (TFA) and Buffer B
being 80% acetonitrile in 0.1% TFA;
said first chain having an amino-terminal sequence beginning with and said second chain having about the formula:
, said protein specifically inhibiting basal secretion of rollicle-stimulating hormone while not inhibiting basal secretion of luteinizing hormone.
elutes from a C8 column of 5 micron particle size and 300 angstrom pore size at between about 27 and about 28 minutes following the instigation of a straight line gradient flow of Buffers A and B, which gradient varies from 25% Buffer B to 90% Buffer B over a period of 45 minutes, at a flow rate of 0.7 ml per minute and a back pressure of about 890 psi, with Buffer A being 0.1% trifluoroacetic acid (TFA) and Buffer B
being 80% acetonitrile in 0.1% TFA;
said first chain having an amino-terminal sequence beginning with and said second chain having about the formula:
, said protein specifically inhibiting basal secretion of rollicle-stimulating hormone while not inhibiting basal secretion of luteinizing hormone.
2. A protein according to Claim 1 having a relative amino acid composition as determined by hydrolysis followed by amino acid analysis in the approximate amounts as follows:
Asx 22, Thr 11, Ser 17, Glx 22, Gly 16, Ala 16, Val 11, Met 5, Ile 8, Leu 22, Tyr 8, Phe 10, His 8, Trp 4, Lys 10, Arg 10, Cys 12, and Pro 23.
Asx 22, Thr 11, Ser 17, Glx 22, Gly 16, Ala 16, Val 11, Met 5, Ile 8, Leu 22, Tyr 8, Phe 10, His 8, Trp 4, Lys 10, Arg 10, Cys 12, and Pro 23.
3. A protein according to either Claim 1 or 2 wherein said first chain is glycosylated.
4. A protein subunit of ovine inhibin having a purity of greater than 90% by weight based upon total protein selected from the group consisting of (a) a first polypeptide having an apparent molecular weight of about 18kD, as measured by gel electrophoresis, having about 135 amino acid residues, and having an amino terminal sequence beginning with , and (b) a second polypeptide having an apparent molecular weight of about 16.5kD, as measured by gel electrophoresis, and having the sequence , wherein the first polypeptide, when combined through disulfide bonding with the second polypeptide, creates a dimer, which dimer elutes from a C8 reverse phase HPLC column of 5 micron particle size and 300 angstrom pore size, at between about 27 and about 28 minutes following the instigation of a straight line gradient flow of Buffers A and B, which gradient varies from 25% Buffer B to 90% Buffer B over a period of 45 minutes, at a flow rate of 0.7 ml per minute and a back pressure of about 890 psi, with Buffer A being 0.1% trifluoroacetic acid (TFA) and with Buffer B being 80% acetonitrile in 0.1% TFA.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US878,063 | 1978-02-15 | ||
| US87806386A | 1986-06-24 | 1986-06-24 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1305829C true CA1305829C (en) | 1992-07-28 |
Family
ID=25371301
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 540422 Expired - Lifetime CA1305829C (en) | 1986-06-24 | 1987-06-23 | Ovine inhibin |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0272309A4 (en) |
| JP (1) | JPH01500121A (en) |
| CA (1) | CA1305829C (en) |
| WO (1) | WO1988000208A1 (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DK0509040T3 (en) * | 1990-01-08 | 1995-04-18 | Genentech Inc | Method for Increasing Fertility in Female Gender |
| DE4000327A1 (en) * | 1990-01-08 | 1991-07-11 | Stefan Heiden | MEDICINES FOR IMPROVING OVARREACTION AND EGG AND EMBRYONE PRODUCTION IN DOMESTIC MUSCLES |
| US5942220A (en) * | 1990-03-16 | 1999-08-24 | Chiron Corporation | Inhibitor of cytokine activity and applications thereof |
| US5658876A (en) * | 1994-04-28 | 1997-08-19 | The General Hospital Corporation | Activin antagonists as novel contraceptives |
| JP2899534B2 (en) * | 1994-12-09 | 1999-06-02 | 全国農業協同組合連合会 | Method of inducing superovulation in cattle |
| US5993820A (en) * | 1996-11-12 | 1999-11-30 | Michigan State University | Chimeric LTB vaccines |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4201770A (en) * | 1973-05-07 | 1980-05-06 | The Ohio State University | Antigenic modification of polypeptides |
| CA1007568A (en) * | 1973-12-13 | 1977-03-29 | Michael C. Attwell | Bovine immunoglobulin isolation process |
| US4268435A (en) * | 1979-02-06 | 1981-05-19 | Research Corporation | Chorionic gonadotropin derived antigen for early pregnancy test and contraceptive vaccine |
| US4409139A (en) * | 1981-08-10 | 1983-10-11 | The Salk Institute For Biological Studies | Gonadostatin |
| WO1986006076A1 (en) * | 1985-04-18 | 1986-10-23 | Biotechnology Australia Pty. Ltd. | Recombinant inhibin |
-
1987
- 1987-06-23 JP JP50410687A patent/JPH01500121A/en active Pending
- 1987-06-23 CA CA 540422 patent/CA1305829C/en not_active Expired - Lifetime
- 1987-06-23 EP EP19870904454 patent/EP0272309A4/en not_active Withdrawn
- 1987-06-23 WO PCT/US1987/001470 patent/WO1988000208A1/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| EP0272309A4 (en) | 1989-12-19 |
| AU7640887A (en) | 1988-01-29 |
| EP0272309A1 (en) | 1988-06-29 |
| WO1988000208A1 (en) | 1988-01-14 |
| AU605162B2 (en) | 1991-01-10 |
| JPH01500121A (en) | 1989-01-19 |
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