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WORLD INTELLECTCAL PROPERTY ORGANIZATION 
International Bureau 




INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification 4 : 

C07K7/06, 7/08,7/10 
C07K 13/00, 15/00 



Al 



(11) International Publication Number: WO 87/ 00528 

(43) International Publication Date: 29 January 1987 (29.01.87) 



(21) International Application Number: PCT/US86/01505 

(22) International Filing Date: 17 July 1986 (17.07.86) 



(31) Priority Application Numbers: 

(32) Priority Dates: 

(33) Priority Country: 



756,866 
784,436 

18 July 1985(18.07.85) 
3 October 1985 (03.10.85) 

US 



(71) Applicant: THE SALK INSTITUTE FOR BIOLOGI- 

CAL STUDIES [USAJS]; 10010 North Torrey Pines 
Road, La JoUa, CA 92037 (US). 

(72) Inventors: LING, Nicholas, Chai-Kwan ; 5324 Bloch 

Street, San Diego, CA 92122 (US). YING, Shao-Yao ; 
3305 Willard Street, San Diego, CA 92122 (US). 
ESCH, Fred, Stephen ; 2929 Fire Mountain Drive, 
#63. Oceanside, CA 92057 (US), GUILLEMIN, Rog- 
er, Charles, Louis ; 7316 Encelia Drive, La Jolla, CA 
92037 (US), 



(74) Agents: WATT, Phillip, H. et al.; Fitch, Even, Tabin & 
Hannery, Room 900, 135 South LaSalle Street, Chi- 
cago, IL 60603 (US). 



(81) Designated States: AT (European patent), AU, BE (Eu- 
ropean patent), CH (European patent), DE (Euro- 
pean patent), FR (European patent), GB (European 
patent), IT (European patent), JP, LU (European pa- 
tent), NL (European patent), SE (European patent). 



Published 

With international search report. 



(54) Title: INHIBIN AND METHOD OF PURIFYING SAME 



(57) Abstract 



Two 32,000-dalton proteins with inhibin activity were isolated from porcine follicular fluid using heparin-Sepharose 
affinity chromatography, followed by gel filtration on Sephacryl S-200 and then four steps of reverse-phase high-perform- 
ance liquid chromatography. Each isolated molecule is composed of two chains having molecular weights of about 18,000 
and about 14,000 daltons, respectively, which are bound together by disulfide bonding. Microsequencing revealed the 
NH2-terminal portion of the 18K chain of both to be Ser-Thr-Ala-Pro-Leu-Pro-Trp-Pro-Trp-Ser-Pro-Ala-Ala-L^u-Arg- 
Leu-Leu-Gln-Arg-Pro-Pro-Glu-GIu-Pro-Ala-Val, of one of the 14K chains to be Gly-Leu-Glu-Cys-Asp-Gly-Arg-Thr-Asn- 
Leu-Cys-Cys-Arg-Gln-Gln-Phe-Phe-Ile-Asp-Phe-Arg-Leu-Ile-Gly-Trp, and of the other I4K chain to be Gly-Leu-Glu-Cys- 
Asp-Gly-Lys-Val-Asn-Ile-Cys-Cys-Lys-Lys-Gln-Phe.Phe-Val-Ser.Phe-Lys-Asp-Ile-Gly.Trp-Asn.Asp-Trp-IIe-Ile-Ala-Pro. 
Both proteins have now been completely characterized, each having a first chain 134 residues long linked by disulfide 
bonding to a second chain 116 or 115 residues long. The first chain is believed to be glycosylated, which accounts for the 
disparity between the number of residues and the apparent molecular weight of 18K. These 32K proteins specifically inhi- 
bit basal secretion of FSH, but not of LH, in a rat anterior pituitary monolayer culture system. The half-maximal ^ff^ctive 
dose of one is 450 pg/ml and of the other is 900 pg/ml. 



FOR THE PURPOSES OF INFORMATION ONLY 



Codes used to identify States party to the PCX on the front pages of pamphlets publishing international appli- 
cations under the PCT. 



AT 


Austria 


GA 


Gabon 


MR 


Mauritania 


AU 


Australia 


GB 


United Kingdom 


MW 


Malawi 


BB 


Barbados 


HU 


Hungary 


NL 


Netheriands 


BE 


Belgium 


IT 


Italy 


NO 


Norway 


BG 


Bulgaria 


JP 


Japan 


RO 


Romania 


BR 


BrazU 


KP 


Democratic People's Republic 


SD 


Sudan 


CF 


Central African Republic 




of Korea 


, SE 


Sweden 


CG 


Congo 


KR 


Republic of Korea 


SN 


Senegal 


CH 


Switzerland 


LI 


Liechtenstein 


SU 


Soviet Union 


CM 


Cameroon 


LK 


Sri Lanka 


TD 


Chad 


DE 


Germany. Federal Republic of 


LU 


Luxembourg 


TG 


Togo 


DK 


Denmark 


MC 


Monaco 


US 


United States of America 


Fl 


Finland 


MG 


Madagascar 






FR 


France 


ML 


Mali 







wo 87/00528 



PCT/US86/0150S 



-1- 

INHIBIN AND METHOD OF PURIFYING SAME 
The present invention relates to a protein 
having inhibin activity isolated substantially to 
homogeneity from bodily material obtained from porcine 

5 animals. The invention also relates to a method of 
purifying porcine inhibin. 

BACKGROUND OF THE INVENTION 
The concept of inhibin as a water-soluble 
substance produced in the gonad but acting specifically 

0 at the pituitary level to inhibit the secretion of 
follicle-stimulating hormone (FSH) was postulated by 
McCullagh in 1932 ( Science , 76 , 19-20). Such 
preferential regulation of the gonadotropin secretion 
has generated a great deal of interest and has prompted 

5 many laboratories in the past fifty years to attempt to 
isolate and characterize this - substance from extracts of 
testis, spermatozoa, rete testis fluid, seminal plasma* 
and ovarian follicular fluid, using various bioassays. 
Although many reports have appeared in' the literature 

0 claiming the purification of inhibin-like material with 
molecular weights ranging from 5,000 to 100,000 daltons, 
subsequent studies have shown that these substances were 
either not homogenous or did not have the high specific 
activity expected of true inhibin (de Jong, Molecular & 

^ Cellular Endocrin. , 13, 1-10 (1979)). Materials having 
inhibin activity may be used to regulate fertility in 
mammalian animals, particularly male animals. 

SUMMARY OF THE INVENTION 
In accordance with the present invention, two 

0 proteins both having a molecular weight of about 32,000 
daltons and having inhibin activity have been 
successfully isolated from porcine follicular fluid. 
These two proteins have been completely characterized 
using microsequencing and molecular biological methods. 



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15 



-2- 

The proteins were isolated to substantial 
homogeneity from material obtained from the bodies of 
porcine animals and are hereinafter referred to as 
Protein A and Protein B. Each protein has a molecular 
weight of about 32,000 daltons (32K) and is composed of 
two polypeptide chains having; molecular weights of 18,000 
and 14,000 daltons, respectively, the chains being linked 
together in the biologically active protein by disulfide 
bonding. Microsequencing showed that the amino-terminal 
amino acid residue sequence of the 18,000 dalton (ISK) 
chain of both proteins was Ser-Thr-Ala-Pro-Leu-Pro-Trp- 
Pro-Trp-Ser-Pro-Ala-Ala-Leu-Arg-Leu-Leu-Gln-Arg-Pro-Pro- 
Glu-Glu-Pro-Ala-Val and that the first six residues of 
both amino-terminals of the 14,000 dalton (14K) chains 
were the same, namely -Gly-Leu-Glu-Cys-Asp-Gly. It was 
also ascertained that the first ten residues of the 14K 
chain of Protein B were Gly-Leu-Glu-Cys-Asp-Gly-Arg-Thr- 
Asn-Leu. Proteins A and B have now been completely 
characterized as a result of further microsequencing and 
the use of molecular biological techniques. Each 32K 
protein exhibits inhibin activity in that it specifically 
inhibits basal secretion of FSH but does not inhibit 
basal secretion of luteinizing hormone (LH). 
The individual- chains are not biologically active. 

Purification of porcine 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 heparin-Sepharose 
affinity chromatography, gel filtration and reverse- 
phase, high-performance liquid chromatography (RP-HPLC). 
BRIEF DESCRIPTION OF THE DRAWINGS 
FIGURE 1 shows chroma tograms representing 
initial steps of purifications of inhibin proteins from 
porcine follicular fluid (PFF) under conditions 
described as follows: 

(a) Heparin-Sepharose affinity chromatography 
of PFF. The inhibin protein was eluted with 1 M NaCl in 
0.01 M Tris-HCl, pH 7. 



20 



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-3- 

(b) Sephacryl S-200 gel filtration of the PFP 
inhibin proteins. The effluent fractions indicated by 
the solid bar near the bottom of Figure la were pooled, 
dialyzed and then divided into eight equal portions for 
this gel filtration. The profile from one of the 
columns is indicative of each column and is shown. 

(c) RP-HPLC purification of the inhibin 
proteins recovered from gel filtration. The components 
of the active region from the gel filtration, as 
determined by in vitro bioassay and designated by the 
solid bar in Figure lb, were pooled, lyophilized and, 
after dissolving in 0.2 N acetic acid, applied directly 
onto a Vydac C4 column and eluted with the indicated 
gradient of acetonitrile in the TEAP buffer system at 9 

15 ini/3 min. Two inhibin proteins. Proteins A and B, 

indicated by the solid bars in Figure Ic, were recovered. 

FIGURE 2 shows chromatograms of RP-HPLC 
purification of inhibin Protein B as follows: 

(a) The active fractions designated by the 
solid bar under B in Fig. Ic were pooled and, after 
being diluted to 3 times their original volume, applied 
directly onto a Vydac C4 column and eluted with the 
indicated gradient of acetonitrile in trif luoroacetic 
acid (TFA) buffer system at 9 ml/3 min. 

(b) The active material denoted by the solid 
bar in Figure 2a was pooled, diluted to 3 times its 
original volume and likewise purified on a Vydac Phenyl 
column with the indicated gradient of acetonitrile in 
triethylammonium phosphate (TEAP) buffer system at 2 

30 ml/2 min. 

(c) The active material accumulated from a 
number of columns identical to that represented by the 
chromatogram of Figure 2b was pooled and concentrated on 
an Aquapore RP-300 column with the indicated gradient of 
acetonitrile in the TFA buffer system at 0.5 ml/min. 

FIGURE 3 shows actual electrophoretic results 
of sodium dodecylsulfate-polyacrylamide gel 



20 



25 



35 



wo 87/00528 PCT/US86/01505 

-4- 

electrophoresis (SDS-PAGE) analysis of the purified 
inhibin Protein B as follows: 

(a) Analysis of Protein B under non-reducing 
condition. 

5 (b) Analysis of Protein B under reducing 

condition. The molecular weight standards are indicated 
on the left. 

FIGURE 4 shows dose-response curves of the 
purified inhibin Protein B (0), as well as the crude PFF 

10 standard ) on the basal secretion of FSH and LH from 
rat anterior pituitary cell cultures. The crude PFF 
standard was a charcoal-stripped and 40% saturated 
ammonium sulfate precipitate of PFF, Schwartz, N. et al. 
Proc. Natl. Acad. Sci . USA 74 5721-5724 ( 1977 ) . 

15 FIGURE 5 shows chroma tograms of RP-HPLC 

purification of inhibin Protein A as follows: 

(a) The active fractions designated by the 
solid bar under A in Fig. Ic were pooled and, after 
being diluted to 3 times their original volume* applied 

20 directly onto a Vydac C4 column and eluted with the 

indicated gradient of acetonitrile in trif luoroacetic 
acid (TFA) buffer system at 9 ml/3 min. 

(b) The active material denoted by the solid 
bar in Figure 5a was pooled, diluted to 3 times its 

25 original volume and likewise purified on a Vydac Phenyl 
column with the indicated gradient of acetonitrile in 
triethylammonium phosphate (TEAP) buffer system at 2 
ml/2 min. 

(c) The active material accumulated from a 

30 number of columns identical to that represented by the 

chromatogram of Figure 5b was pooled and concentrated on 
an Aquapore RP-300 column with the indicated gradient of 
acetonitrile in the TFA buffer system at 0.5 ml/min. 
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 

35 Using a multi-step procedure # two 32,000 dalton 

peptides were isolated to substantial homogeneity from 
porcine follicular fluid (PFF). Each protein (Protein A 



wo 87/00528 



PCT/US86/01505 



10 



15 



20 



25 



30 



35 



-5- 

and Protein B) is composed of two chains of 18K and 14K, 
respectively, and the chains of the intact molecule are 
held together by disulfide bonding, the linkage between 
the chains being necessary for biological activity. An 
amino acid analysis of the total protein has been 
performed for each, and the amino acid residue sequence 
of the amino-terminus of each chain has been determined 
by microsequencing. The amino-terminal sequence of the 
18K chain of each protein was found to be: Ser-Thr-Ala- 
Pro-Leu-Pro-Trp-Pro-Trp-Ser-Pro-Ala-Ala-Leu-Arg-Leu-Leu- 
Gln-Arg-Pro-Pro-Glu-Glu-Pro-Ala-Val. As a result of 
further work, it is believed that both 18K chains have 
exactly the same sequence of 134 amino aCid residues and 
have a free acid C-terminus. 

Microsequencing initially showed that the 
araino-terminal sequences of the 14K chains of the first 
six residues of both chains were the same and that the 
first ten residues of the 14K chain of Protein B were 
Gly-Leu-Glu-Cys-Asp-Gly-Arg-Thr-Asn-Leu . Further 
sequencing showed that the. next* 15 residues were 
^ll"^12~^^^"^^^"^-^^~^^®"^^®"^^®"-^sP"^^^"Arg-Leu- 
^23"^^^"'^^^' ^^^^ ^11 possibly being Ser, with X^^ 
likely being Cys and with X^j being lie or Leu. Later 
it was determined that both X^^^ and X^^ were Cys and 
that X^j was lie. The ability to sequence the chains 
demonstrates that the protein has been purified to at 
least about 90% by weight of total protein as do the 
sharp elution peaks of the protein in the final 
chromatographic purification step. 

The C-termini of all three chains are free acid. 

Further microsequencing showed that the N-terminus of 

the 14K chain of Protein A has the following sequence: 

Gly-Leu-Glu-Cys-Asp-Gly-Lys-Val-Asn-Ile-Cys-Cys-Lys-Lys- 

Gln-Phe-Phe-Val-Ser-Phe-Lys-Asp-Ile-Gly-Trp-Asn-Asp-Trp- 
Ile-Ile-Ala-Pro. 

Protein A has now been completely characterized 
and found to include a 134-residue chain (the ISK chain) 



wo 87/00528 



PCT/US86/01505 



-6- 

that is linked by one or more disulfide bridges to a 
116-residue chain (the 14K chain) which appears to 
include at least one internal disulfide bridge. 
Protein B has the same 18K chain linked to a homologous 
5 14K chain. 

Each 32K protein is acidic, having a pKa of 
4.8, and is generally soluble in aqueous media. Each is 
glycosylated to a limited extent, as determined by 
limited affinity to concanavalin A. Each 32K protein 

10 exhibits inhibin activity in that it specifically 
inhibits basal secretion of FSH in a rat anterior 
pituitary monolayer culture system. Protein B exhibits 
a half-maximal effective dose of 450 pg/ml, as 
calculated by the computer program BIOPROG, and Protein 

15 A exhibits an ED^^ of 900 pg/ml. Each 32K protein is 
useful for regulating fertility of mammalian animals, 
particularly males. 

Each 32K protein has apparent similarities to a 
56,000 dalton (56K) protein isolated from bovine 

20 follicular fluid, a protein composed of two chains of 
molecular weights 44,000 and 14,000 daltons (44K and 
14K) bound together by disulfide bonds. The first three 
amiho-terminal residues of the 44K bovine chain reported 
by Robertson et al.. Bio. Chem. Biophys. Res> Commun. 

25 176 220-226 (1985) are different from either of the two 
chains of either of the 32K porcine proteins ^hat were 
isolated, but the second and third residues of the 14K 
chain of the 56K bovine peptide are identical to those 
of the 14K chain of both of the 32K porcine peptides. 

30 The full relationship, if any, between the 3 2K porcine 

proteins and the S6K bovine protein is not known at this 
time. 

Each 32K protein is definitely different from 
the "alpha-inhibins" Ramasharma, K. et al. Science 223 , 
35 1199-1201 (1984); Li, C. H. et al., Proc. Natl. Acad. 

Sci. USA 82 4041-4044 (1985) and "beta-inhibin" Seidah, 
N. FEES Letter 175 349-354 (1985) recently isolated and 



wo 87/00528 



PCT/US86/0150S 



-7- 

characterized from human seminal plasma, 
"Alpha-inhibins" are reported to be single-chain 
polypeptides all derived from a 92-amino-acid-residue 
parent molecule. However, neither a synthetic replicate 
5 of the 31-amino-acid NH2-terminal fragment of 

alpha-inhibin-52 nor the native product inhibited the 
basal FSH-releaslng activity in the rat anterior 
pituitary cell culture, Yamashiro, D. et al., Proc. 
Natl. Acad. Sci . USA 81 5349-5402 (1984). "Beta-Inhibin" 

10 is described as a single-chain polypeptide with 94 amino 
acid residues, with the bioactive core allegedly 
residing at the COOH-terminal 67-94 fragment, Arbatti,N. 
et al. FEES Letters , 181 , 57-63 (1985). This fragment 
has been synthesized, but it is found to be inactive, up 

15 to a concentration of 5 pg/ml, to inhibit the basal 
secretion of FSH in the rat anterior pituitary cell 
culture assay. The bioassay methods (for "inhibin 
activity") used in these two laboratories, Ramasharraa et 
al. supra, and Li et al. supra . , are substantially 

20 different from those described herein. 

In an inhibin purification procedure according 
to the invention, porcine inhibin is isolated from crude 
extract material obtained from a porcine animal body, 
specifically porcine follicular fluid (PFF) although 

25 other appropriate bodily extracts might be us-ed, by 
successive purification procedures that include 
heparin-Sepharose affinity chromatography, gel 
filtration and at least one and preferably several 
RP-HPLCs of different conditions of stationary phase 

30 and/or mobile phase. The same procedure is useful in 
obtaining a desired mammalian inhibin protein from a 
crude extract resulting from recombinant DNA processes. 

In the preferred procedure by which porcine 
inhibin was first isolated to substantial purity, PFF 

35 was first purified by heparin-Sepharose affinity " 

chromatography, next by gel filtration on Sephacryl 
S-200 gel and then with four successive RP-HPLCs using 



different mobile phase gradients and/or derivatized 
silica supports • Preferably stationary phases having 
relatively low hydrophobicity are used, with C3-C8 
columns being preferred and C3-C5 and phenyl columns 
being particularly preferred. Solute specificity of the 
mobile phase is preferably adjusted by varying the 
concentration of an organic component, particularly 
acetonitrile. Although a single RP-HPLC fractionation 
significantly increases the purity relative to the gel- 
filtrated material, two or morer and preferably four, 
RP-HPLC purifications are generally performed subsequent 
to sucessive treatment by heparin-Sepharose 
chromatography and gel filtration. 

The starting material for the procedure was 
frozen PFF that was procured from J.R. Scientific, 
Woodland, CA. Approximately 18 liters of such frozen 
PFF. were processed in 250 ml batches to isolate the 
inhibin product. The first step of the purification was 
heparin-Sepharose affinity chromatography, in which the 
protein is adsorbed to the Sepharose-bound heparin 
moieties under application conditions, and the adsorbed 
inhibin material is recovered by IM NaCl elution. This 
step greatly expedites the purification procedure for 
crude extracts because it allows a relatively large 
volume of a crude extract, such as PFF, to be processed 
fairly rapidly while recovering an amount of protein 
exhibiting total inhibin activity equal to at least 90% 
of that of the curde extract. 

Throughout the purification procedure, the 
inhibin bioactivity was monitored by an in vitro 
bioassay using rat anterior pituitary monolayer culture. 
Vale, W. et al. Endocrinology , 91_' 562-572 (1972). In 
brief, 21-day-old female rat anterior pituitaries are 
collected, enzymatically dispersed and plated in 1-0% 
fetal bovine serum in HDMEM (GIBCO Laboratories, Santa 
Clara, CA. ) into 24-well tissue culture plates (Falcon 
Plastic, Oxnard, CA. ) on day 1. On day 2, the medium is 



wo 87/00528 



PCT/US86/01505 



10 



-9- 

changed to 1% fetal bovine serum in HDMEM, and the 
sample is added. Incubation is continued for another 48 
hours. The medium is then harvested, and the LH and PSH 
contents are determined by radio-immunoassay (RIA) using 
materials provided by The Pituitary Hormone Program of 
NIADDKD, In this assay, the inhibin proteins inhibit 
the basal release of FSH only but not that of LH, as 
compared to control cells that receive the incubation 
medium only. 

For the detection of inhibin activity in the 
various column fractions, aliquots ranging from 0.01% to 
0.1% by volume were removed, and after adding 100 pg 
human serum albumin in 100 pi water, the solvents were 
evaporated in a Speed-Vac concentrator (Savant, 
^5 Hicksville, N.Y.). The residue was redissolved in 3 ml 
1% fetal bovine serum in HDMEM, filtered through a 
Millex-GS 0.22 um filter (Millipore Corp., Bedford, MA) 
and assayed in duplicate. To speed up the bioassays 
during the purification process, only basal inhibition 
of FSH secretion exerted by the inhibin activity was 
determined and plotted in the region where the inhib)in 
proteins were expected to migrate in the chromatograms . 

To perform the heparin-Sepharose affinity 
chromatography, a 500 ml bottle of frozen PFF was 
defrosted, and the cell debris were spun down in a 
Beckman J2-21 centrifuge (Beckman Instruments, Inc., 
Palo Alto, CA. ) using a JA-20 rotor at 10,000 rpm for 30 
minutes. One half of the supernatant (250 ml) was 
diluted to 10 times its volume by the addition of 2,250 
ml of 0.01 M Tris-HCl containing 0.1 M NaCl, pH 7, in a 
4 liter Erlenmeyer flask and pumped simultaneously via 
eight silastic tubes (0.7^mm ID) into eight 
heparin-Sepharose (Pharmacia Fine Chemicals, Piscataway, 
N. J.) columns (3.5 x 9 cm) by two Rabbit 4-chanTiel 
peristaltic pumps (Rainin Instrument Co., Inc., 
Emeryville, CA. ) at 40 ml/hr per column. After all the 
fluid had been pumped through the heparin-Sepharose, the 



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PCT/US86/01505 



-10- 

eight columns were washed simultaneously with 3.5 liters 
of 0,01 M Tris-HCl, pH 7, containing 0.1 M NaCl in the 
same manner. The adsorbed proteins with inhibin 
activity were removed by washing the eight columns 
5 simultaneously with 1.3 liters of 0.01 M Tris-HCl 

containing 1 M NaCl, pH 7, as above, and the wash was 
collected into fractions of 16 ml. Figure la shows a 
typical elution profile of the inhibin activity material 
from PFF. The inhibin activity was monitored by the iii 
10 vitro bioassay described above* The columns were 

regenerated by further washing with 1.6 liters 2M NaCl 
in 0.01 M Tris-HCl, pH 7, and re-equilibrat^d with 3.5 
liters 0.01 M Tris-HCl containing 0.1 M NaCl for 
purification of the remaining 250 ml of PFF. 
15 Next, the material was fractionated by gel 

filtration to separate proteins generally according to 
their molecular weights. The fractions having inhibin 
activity extracted by the eight heparin-Sepharose 
columns were pooled (400 ml) and dialyzed overnight to 
20 remove NaCl in a 28.6 mm cylinder diameter Spectrapor 
No. 3 membrane tubing with cutoff at 3,500 
(Spectrum Medical Industries, Inc., Los Angeles, CA. ) 
against 16 liters of 30% acetic acid. The retained 
fluid was centrifuged, as above, to remove a white 
25 precipitate, and the supernatant was divided into eight 
equal portions for applying to ei^ht 5 x 100 cm 
Sephacryl 3-200 superfine columns (Pharmacia Fine 
Chemicals, Piscataway, N. J.). Each column was elut^d 
with 30% acetic acid at 20 ml for 22 min., and the 
column fractions were monitored by UV absorption at 280 
nm and by bioassay. 

Fig. lb shows the elution profile of the 
inhibin material purified in a Sephacryl S-200 column. 
Two elution zones of approximately equal amounts of 
activity were detected, one eluting at about 30,000 
and the other at about 12,000. However, based upon 
the protein concentration as determined by UV 



30 



35 



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PCT/LS86/01S05 



10 



-11- 

absorption, the low-molecular-weight zone contains much 
higher specific activity. As a result, this region was 
selected for further purification by RP-HPLC. 

The low-molecular-weight inhibin protein from 
the eight S-200 columns was pooled and lyophilized. The 
lyophilized material (40 mg) was dissolved in 40 ml 0.2N 
acetic acid and filtered through a Millex-HA 0.45 pm 
filter (Millipore Corp., Bedford, MA.). The filtrate 
was applied directly onto a 1 x 25 cm Vydac 5-pm 
particle-size C4 column (The Separations Group, 
Hesperia, CA. ) and developed with a gradient of TEAP 
buffer as shown in Figure Ic. In the TEAP system, 
buffer A consists of 0.25 N. tri ethyl ammonium phosphate, 
pH 3, and buffer B is 80% acetonitrile in buffer A. 
15 After all the filtrate had been loaded, the column was 

washed with the aqueous buffer A until the UV absorption 
reached baseline. The fractions exhibiting inhibin 
activity were separated in a Beckman 332 gradient liquid 
chromatography system (Beckman Instruments, Inc., 
20 Berkely, CA. ) equipped with a Spectroflow 757 UV 

detector (Kratos Analytical Instruments, Ramsey, N. J.), 
a Soltec 220 recorder (Soltec Corp., Sun Valley, CA. ) 
and a Redirac 2112 fraction collector (LKB Instruments, 
Inc., Gathersburg, MD. ) . Two zones of substantial 
25 inhibin activity were detected, with more activity 

exhibited by the earlier eluting zone (inhibin Protein 
A) than the later eluting zone (inhibin Protein B), s-ee 
Figure Ic. 

Inhibin Protein B from the various columns were 
pooled and further purified by two more RP-HPLC steps. 
The first step uses a 1 x 25 cm Vydac 5-pm-particle-size 
C4 column and a trif luoroacetic acid (TFA) buffer system 
(Figs. 2a) and the second step employs a 1 x 25 cm Vydac 
5-|im-particle-size Phenyl column and the TEAP buffer 
system, as shown in Fig. 2b. In the TFA system, buffer 
A contains 1 ml trif luoracetic .acid in 999 ml water and 
buffer B is 1 ml trif luoroacetic acid in 199 ml water 



30 



35 



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PCT/US86/0150S 



-12- 

and 800 ml acetonitrile. Finally, inhibin Protein B 
accumulated from a few batches was concentrated by 
RP-HPLC using a 0.46 x 25 cm Aquapore RP-300 10 
um-particle-size column (Brownlee Labs., Santa Clara, 
5 CA. ) and the TFA buffer system as shown in Fig. 2c. 
Altogether, a total of approximately 60 pg of inhibin 
Protein B was purified from the 18 liters of PFF. 

Inhibin Protein A from the various columns was 
pooled and further purified by two more RP-HPLC steps. 

10 The first step uses a 1 x 25 cm Vydac 5-pm-particle-size 
C4 column and a * trif luoroacetic acid (TFA) buffer system 
(Figs. 5a) and the second step employs a 1 x 25 cm Vydac 
5-pm-particle-size Phenyl column and the TEAP buffer 
system, as shown in Fig. 5b. In the TFA system, buffer 

15 A contains 1 ml tri f luoracetic acid in 999 ml water and 
buffer B is 1 ml trif luoroacetic acid in 199 ml water 
and 800 ml acetonitrile. Finally, inhibin Protein A 
accumulated from a few batches were concentrated by 
RP-HPLC using a 0.46 x 25 cm Aquapore RP-300 10 

20 pm-particle-size column (Brownlee Labs., Santa Clara, 
CA. ) and the TFA buffer system as shown in Fig. 5c. 
Altogether, a total of approximately 600 pg of inhibin 
Protein A was purified from the 18 liters of PFF. 

Amino acid analyses of the substantially 

25 homogeneous inhibin Proteins A and B were performed as 
described in Bohlen P., et al. Anal. Biochem . 126 
144-156 (1982), and the results are shown in Table 1 
below. 



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10 



15 



20 



-13- 

TABLE 1 

AMINO ACID COMPOSITION OP PURIFIED INHIBIN 
PROTEIN FROM PORCINE FOLLICULAR FLUID 

Amino Acid Protein A* Protein B* 



A e Y 






1 • D 


zl .U 


+ 1.1 


TTV» V- 

1 nr 


11 • D 


1 

T 


A C 


13.5 


-l_ A ^ 

+ 0.5 


Ser 


16 .3 


+ 


1.4 


18.1 


+ 0.7 




21.3 


+ 


1.4 


22 . 5 


+ 0.5 


vjxy 


ly • y 


.1 

T 


1.9 


22.4 


+ 0.7 




19 • 7 


+ 


1.4 


22 .0 


+ 0.6 


vai 


n /I T 
14.1 


+ 


1.8 


14.4 


+ 0.4 


Met 


6.1 


+ 


0.4 


5.8 


+ 0.3 


He 


14.6 


+ 


1.7 


10.3 


+ 0.2 


Leu 


27.3 




2.1 


27.8 


+ 0.8 


Tyr 


10.7 


+ 


1.3 


11.4 


+ 0.4 


Phe 


11.6 


+ 


1.4 


10.0 


+ 0.2 


His 


14.1 


+ 


1.2 


7.9 


+ 0.6 


Trp 


4.4 




0.3 


3.9 


+ 0.4 


Lys 


11.9 


+ 


0.9 


5.4 


+ 0.2 


Arg 


13.9 


+ 


0.6 


15.8 


+ 0.4 


Cys** 


14.4 


+ 


0.2 


14.2 


+ 0.9 


Pro 


29.2 


+ 


0.9 


29.6 


+ 1.1 



Data corresponds to the mean + SD of four analyses 
and normalized to a protein of 32,000 daltons. 



25 ** Cysteine was determined as cysteic acid after 
performic acid oxidation. 

The inhibin Protein B from the final RP-HPLC 
purification was analyzed under reducing and 
non-reducing conditions in 1-mm-thick 15% acrylamide gel 

30 according to the method of Laemmli, V., Nature 227 
680-685 (1970). The protein was revealed by silver 
staining reagent (BIO-RAD, Richmond CA. ) . The following 
molecular weight standards were used to calibrate the 
gel: bovine serum albumin (M^ "67,000), ovalbumin 

35 (M^=43,000), alpha-chymotrypsinogen (M^= 25,700) and 



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-14- 

lysozyme (M^= 14,500). In non-reducing condition, 2 
Jig of inhibin protein in 20 pi water was incubated with 
20 |il sample buffer (0.152 M Tris-HCl, pH 6.8, . 
containing 20% glycerol (V/V) , 4% sodium dodecylsulfate 
5 and 0.04% bromphenol blue) for 1 hour at 37*C .prior to 
loading onto the gel. The electrophoresis was performed 
at a constant 200 volts for 6 hours at room 
temperature. In reducing condition, 2 ug of protein was 
incubated, first with 20 pi of 0.02 M dithiothreitol for 

10 15 min at 37*'C and then 20 pi of sample buffer was added 
and the incubation continued for one more hour before 
the sample was applied to the gel. Electrophoresis was 
carried out as above, except that 0.005 M dithiothreitol 
was included in the electrophoretic buffer. On SDS-PAGE 

15 under non-reducing condition, inhibin Protein B showed a 
single band migrating at 32,000 (See Figure 3a). 
Under reducing condition, inhibin Protein B separated 
into two bands, one migrating at 18,000 and the 
other 14,000 (See Figure 3b). Similar 

20 electrophoresis of Protein A showed two such bands plus 
one at M 8,000 which was felt to possibly indicate 
the presence of impurity. 

NH2-terminal sequence analyses of the 18K and 
14K chains of 32K inhibin Proteins A and B were 

25 accomplished by first separating the two chains by 
SDS-PAGE under reducing conditions, followed by 
electro-blotting of the separated protein chains onto 
GF/C paper which was subsequently punched out for direct 
microsequencing. Briefly, each inhibin protein (12-15 

30 yg) was electrophoresed on 0 .5-mm-thick 15% acrylamide 
gel (8 X 10 cm) under reducing condition, as described 
above. After electrophoresis , the gel was immediately 
washed 3 times with 0.5% NP-40 (Sigma Chemical Co., St. 
Louis, MO.) in 0.5% acetic acid at room temperature. 

35 The gel was then placed between 2 sheets of 

tr if luoracetic acid-activated GF/C paper (Whatman, 
Clifton, N.J.) which had been wet briefly with the 



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PCT/US86/01S05 



-15- 

blotting buffer (2% acetic acid containing 0.5% NP-40). 
One more activated GF/C paper was placed on the cathode 
side in case the protein was not completely absorbed by 
the first paper. The outer surface of the GP/C paper 
5 was protected by Whatman No. 3 filter paper, and the 
whole assembly inserted between two Scotch-Brite pads 
held together by a plastic grid (BIO-RAD, Richmond, 
CA). This was electrophoresed in the blotting buffer at 
a constant voltage of 60 V for 16 hours at 4**C to 

10 transfer the positively charged proteins onto the GF/C 
paper. The electrolytically transferred protein chains 
were revealed with Coomassie blue staining. 1.2 cm 
diameter circles were punched out of the stained protein 
bands and subjected directly to microsequencing as 

15 described above. 

Microsequencing, as described in Esch, F. Anal. 
Biochem. 136, 39-47, 1984, of intact inhibin protein 
beginning at the NH^-terminus had consistently 
revealed two residues of approximately equal 

20 concentration at every cycle, indicating each protein is 
composed of two chains. Based upon the results from 
multiple sequencing analyses of both intact and reduced 
inhibin proteins, the NH^- terminal residues of the 16K 
chain of each inhibin protein are Ser-Thr-Ala-Pro-Leu- 

25 Pro-Trp-Pro-Trp-Ser-Pro-Ala-Ala-Leu-Arg-Leu-Leu-Gln- 
Arg-Pro-Pro-Glu-Glu-Pro-Ala-Val. The NHj-terminal 
residues of the 14K chain of inhibin Protein B are 
Gly-Leu-Glu-Cys-Asp-Gly-Arg-Thr-Asn-Leu^Cys-Cys-Arg- 
Gln-Gln-Phe-Phe-Ile-Asp-Phe-Arg-Leu-Ile-Gly-Trp- , and 

30 the NH^-terminal residues of the 14K chain of inhibin 
Protein A are Gly-Leu-Glu-Cys-Asp-Gly-Lys-Val-Asn-Il«- 
Cys-Cys-Lys-Lys-Gln-Phe-Phe-Val-Ser-Phe-Lys-Asp-Ile- 
Gly-Trp-Asn-Asp-Trp-Ile-Ile-Ala-Pro-. 

Once a substantial portion of the sequence of 

35 the three chains of inhibin protein are known, the mRNA 
encoding the chains can be isolated, and the <:DNA's can 
be synthesized by recombinant DNA techniques. Messenger 



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•16- 

RNA (mRNA) is obtained from ovarian follicules 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 
5 suitable host to create a cDNA library. 

Based upon the known partial amino acid residue 
sequences of the three inhibin chains, labelled 
oligonucleotides are synthesized for detecting cDNA 
corresponding to each chain. Because of the degeneracy 

10 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 immunological expression assay with 

15 antibody raised against inhibin or one of the inhibin 

chains. Immunological expression assay may also be used 
to confirm screening with hybridization probes. 

From selected clones, cDNA is excised and 
inserted into appropriate vectors under €he control of 

20 promoter sequences, and the- vectors are transformed into 
cell lines for expression of the recombinant inhibin 
chains. Although vectors containing the genes for an 
appropriate pair of chains could . conceivably be 
transformed into the same cell line, for simplicity, 

25 vectors for expression of each chain are preferably 

transformed separately into cell lines. The individual 
inhibin chains can then be isolated from the cellular 
material and/or the cell culture medium and are useful 
as intermediates for the preparation of the bioactive 

30 inhibin. Appropriate quantities of the 18K and the 14K 
chains are then subjected to oxidizing conditions which 
promote disulfide bonding between the chains to produce 
inhibin. 

The foregoing molecular biology techniques may 
35 also be used to read the gene sequences encoding the 
separate inhibin chains, and thereby completely 
characterize the protein chains. Protein A has now been 



-17- 

completely characterized and found to include a 
134-residue chain (the 18K chain) that is linked by one 
or more disulfide bridges to a 116-residue chain (the 
14K chain). The 134-residue chain has the following 
formula: H-Ser-Thr-Ala-Pro-Leu-Pro-Trp-Pro-Trp-Ser-Pro- 
Ala-Ala-Leu-Arg-Leu-Leu-Gln-Arg-Pro-Pro-Glu-Glu-Pro-Ala- 
Val-His-Ala-Asp-Cys-His-Arg-Ala-Ser-Leu-Asn-Ile-Ser-Phe- 
Gln-Glu-Leu-Gly-Trp-Asp-Arg-Trp-Ile-Val-His-Pro-Pro-Ser- 
Phe-Ile-Phe-His-Tyr-Cys-His-Gly-Gly-Cys-Gly-Leu-Pro-Thr- 
Leu-Pro-Asn-Leu-Pro-Leu-Ser-Val-Pro-Gly-Ala-Pro-Pro-Thr- 
Pro-Val-Gln-Pro-Leu-Leu-Leu-Val-Pro-Gly-Ala-Gln-Pro-Cys- 
Cys-Ala-Ala-Leu-Pro-Gly-Thr-Met-Arg-Ser-Leu-Arg-Val-Arg- 
Thr-Thr-Ser-Asp-Gly-Gly-Tyr-Ser-Phe-Lys-Tyr-Glu-Thr-Val- 
Pro-Asn-Le,u-Leu-Thr-Gln-His-Cys-Ala-Cys-Ile-OH. The 
116-residue chain has the following formula: H-<jly-Leu- 
Glu-Cys-Asp-Gly-Lys-Val-Asn-Ile-Cys-Cys-Lys-Lys-Gln-Phe- 
Phe-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. This characterization is in agreement with 
the earlier analyses of the purified protain material 
obtained from PFF; the disparity between the number of 
residues in the first chain and the measured value of 
18K is accounted for by the presence of the 
aforementioned glycosylation. It is believed that a 
carbohydrate moiety having a molecular weight of about 
3000 daltons is attached to the side chain of the Asn 
residue in the 36-position of the first chain. 

Protein B has also been characterized and the 
18K chain is believed to have the same sequence as 
Protein A. The 14K chain has been found to have the 
following 115-residue formula: H-Xjly-Leu-tjlu-Cys-Asp-Gly- 
Arg-Thr-Asn-Leu-Cys-Cys-Arg-^ln-Gln-Phe-Phe-Ile-Asp-Phe^ 



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PCT/US86/01S05 



-18- 

Arg-Leu-Ile-Gly-Trp-Ser-Asp-Trp-Ile-Ile-Ala-Pro-Thr-Gly- 
Tyr-Tyr-Gly-Asn-Tyr-Cys-Glu-Gly-Ser-Cys-Pro-Ala-Tyr-Leu- 
Ala-Gly-Val-Pro-Gly-Ser-Ala-Ser-Ser-Phe-His-Thr-Ala-Val- 
Val-Asn-Gln-Tyr-Arg-Met-Arg-Gly-Leu-Asn-Pro-Gly-Thr-Val- 
5 Asn-Ser-Cys-Cys-Ile-Pro-Thr-Lys-Leu-Ser-Thr-Met-Ser-Met- 
Leu-Tyr-Phe-Asp-Asp-Glu-Tyr-Asn-Ile-Val-Lys-Arg-Asp-Val- 
Pro-Asn-Met-Ile-Val-Glu-Glu-Cys-Gly-Cys-Ala-OH- This 
characterization is in agreement with the earlier 
analyses of the purified protein material obtained from 
10 pFF. 

The invention thus provides polypetides or 
relatively short proteins between about 115 residues and 
about 134 residues in length which are useful as 
intermediates to create disulf ide-bonded dimers that are 

15 biologically active. More specifically, the invention 
provides the three polypeptides set, forth as follows: 
(a) H-Ser-Thr-Ala-Pro-Leu-Pro-Trp-Pro-Trp-Ser-Pro-Ala- 
Ala-Leu-Arg-Leu-Leu-Gln-Arg-Pro-Pro-Glu-Glu-Pro-Ala-Val- 
His-Ala-Asp-Cys-His-Arg-Ala-Ser-Leu-Asn-Ile-Ser-Phe-Gln- 

20 Glu-Leu-Gly-Trp-Asp-Arg-Trp-Ile-Val-His-Pro-Pro-Ser-Phe- 
Ile-Phe-His-Tyr-Cys-His-Gly-Gly-Cys-Gly-Leu-Pro-Thr-Leu- 
Pro-Asn-Leu-Pro-Leu-Ser -Val-Pro-Gly-Ala-Pr o-Pro-Thr -Pro- 
Val-Gln-Pro-Leu-Leu-Leu-Val-Pro-Gly-Ala-Gln-Pro-Cys-Cys- 
Ala-Ala-Leu-Pro-Gly-Thr-Met-Arg-Ser-Leu-Arg-Val-Arg-Thr- 

25 Thr-Ser-Asp-Gly-Gly-Tyr-Ser-Phe-Lys-Tyr-Glu-Thr-Val-Pro- 
Asn-Leu-Leu-Thr-Gln-His-Cys-Ala-Cys-Ile-OH; (b) H-Gly- 
Leu-Glu-Cys -Asp-Gly-Arg -Thr -Asn-Leu-Cys -Cys -Arg-Gln-<31n- 
Phe-Phe-Ile-Asp-Phe-Arg-Leu-Ile-Gly-Trp-Ser-Asp-Trp-Ile- 
Ile-Ala-Pro-Thr-Gly-Tyr-Tyr-Gly-Asn-Tyr-Cys-<31u-Gly-Ser- 

30 Cys-Pro-Ala-Tyr-LeurAla-Gly-Val-Pro-Gly-Ser-Ala-Ser-Ser- 
Phe-His-Thr-Ala-Val-Val-Asn-Gln-Tyr-Arg-Met-Arg-Gly-Leu- 
Asn-Pro-Gly-Thr-Val-Asn-Ser-Cys-Cys-Ile-Pro-Thr-Lys-Leu- 
Ser-Thr-Met-Ser-Met-Leu-Tyr-Phe-Asp-Asp-<31u-Tyr-Asn-Ile- 
Val-Lys-Arg-Asp-Val-Pro-Asn-j4et-l'le-Val-Glu-Glu-Cys-Gly- 

35 Cys-Ala-OH and (c) H-Gly-Leu-Glu-Cys-Asp-Gly-Lys-Val-Asn 



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PCT/US86/01S05 



-19- 

Ile-Cys-Cys-Lys-Lys-Gln-Phe-Phe-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- 
5 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. For example, 
the 134 -residue polypeptides may be combined with one of 

10 the other shorter polypeptides to form biologically 
active disulphide-bonded heterodimers. 

Substantially pure 32K inhibin or the nontoxic 
salts thereof, combined with a pharmaceutically 
acceptable carrier to form a pharmaceutical composition^ 

15 may be administered to mammals, including humans, either 
intravenously , subcutaneously , pe rcutaneously , 
intramuscularly or orally for control of fertility. 
Administration of inhibin induces decreased fertility in 
^female mammals and decreases spermatogenesis in male 

20 mammals. Administration of a sufficient amount of 

inhibin induces infertility in mammals. Inhibin is also 
useful for tests to diagnose infertility. 

Such peptides are often administered in the 
form of pharmaceutically acceptable nontoxic salts, such 

25 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, 

30 benzoate, succinate , malate , ascorbate, tartrate and the 
like. If the active ingredient is to be administered in 
tablet form, the tablet may contain a binder, such as 
tragacanth, <:orn starch or gelatin; a disintegrating 
agent, such as alginic acid; and a lubricant, such as 

35 magnesium stearate. 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 t)e effected. 



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-20- 

Inhibin should be administered under the 
guidance of a physician, and pharmaceutical compositions 
will usually contain an effective amount of the peptide 
in conjunction with a conventional, pharmaceutically- 
5 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 

10 as a male contraceptive. 

Although the method of purification of inhibin 
has been described primarily in terms of isolation from 
PFF, innibin can be similarly purified from other crude 
extracts. The term "crude extracts" as used herein 

15 refers to other mammalian body material in addition to 
follicular fluid, as well as to extracts from organisms 
including laboratory microorganisms, such as prokaryotes 
(e.g. E. Coli ) and eukaryotes (e.g. S. cerevisiae yeast) , 
wnich have been transformed by means of state of the art 

20 methodology to manufacture mammalian inhibin protein. 

Although the invention has been described with 
regard to its preferred embodiments, which constitute 
tne best mode presently known to the inventors, it 
should be understood that various changes and 

25 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. 

Particular features of the invention are 

30 emphasized in the claims which follow. 



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PCT/US86/01S0S 



-21- 

WHAT IS CLAIMED IS: 

1. A 32,000-dalton protein having a purity of 
at least about 90% by weight of total proteins, said 
protein having a pKa of about 4.8, said protein being 
composed of a first polypeptide chain having a molecular 

5 weight of about 18,000 and a second polypeptide chain 
having a molecular weight of about 14,000 daltons, said 
first chain and said second chain being linked to each 
other through disulfide bonding in the active protein, 
said first chain having an amino-terminal sequence 

10 beginning with Ser-Thr-Ala-Pro-Leu-Pro-Trp-Pro-Trp-Ser- 
Pr o- Ala-Ala-Leu- Arg-Leu-Leu-Gln-Arg-Pro-Pro-Glu-Glu-Pro- 
Ala-Val and said second chain having an amino-terminal 
sequence beginning with Gly-Leu-Glu-Cys-Asp-Gly , said 
protein specifically inhibiting basal secretion of 

15 follicle-stimulating hormone while not inhibiting basal 
secretion of luteinizing hormone. 

2. A protein according to claim 1 wherein 
said second chain has an amino terminal sequence 
beginning with Gly-Leu-Glu-Cys-Asp-Gly-Arg-Thr-Asn-Leu. 

20 3. A protein according to claim 1 wherein 

said second chain has an amino terminal sequence 
beginning with Gly-Leu-Glu-Cys-Asp-<31y-Arg-Thr-Asn- 
Leu-X-Cys-Arg-Gln-Gln-Phe-Phe-Ile-Asp-Phe-Arg-Leu-Ile- 
Gly-Trp, wherein X is an unknown amino acid residue. 

25 4. A protein according to claim 1 wherein 

said second chain has the formula: H-Gly-Leu-Glu-Cys- 

Asp-Gly-Lys-Val-Asn-Ile-Cys-Cys-Lys-Lys-Gln-Phe-Phe-Val- 

Ser-Phe-Lys-Asp-Ile-Gly-Trp-Asn-Asp-Trp-Ile-Ile-Ala-Pro. 



-22- 

5. A protein according to claim 1 wherein 

said second chain has the formula: H-Gly-Leu-Glu-Cys- 
Asp-Gly-Lys-Val-Asn-Ile-Cys-Cys-Lys-Lys-GIn-Phe-Phe-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-Gin-Asn-Met-Ile-Val-Glu-Glu-Cys-Gly-Cys-Ser- 
OH. 

6. A protein according to Claim 1 having an 
amino acid composition of about Asx 21,0, Thr 13.5. Ser 
18.1, Glx 22.5, Gly 22.4, Ala 22.0, Val 14.4, Met 5.8, 
He 10.3, Leu 27.8, Tyr 11.4, Phe 10.0, His 7.9, Trp 
3.9, Lys 5.4, Arg 15.8, Cys 14.2, and Pro 29.6. 

7. A protein according to Claim 1 having an 
amino acid composition of about Asx 18.5, Thr 11.5. Ser 
16.3, Glx 21.3, Gly 19.9, Ala 19.7, Val 14.1, Met 6.1, 
He 14.6, Leu 27.3, Tyr 10.7, Phe 11.6, His 14.1, Trp 
4.4, Lys 11.9, Arg 13.9, Cys 14.4, and Pro 29.2. 

8. A protein according to Claim 1 wherein 
said first chain has the formula: H-Ser-Thr-Ala-Pro-Leu- 
Pr o-Trp-Pro-Tr p-Ser -Pro-Ala-Ala-Leu- Arg -Leu-Leu-Gln -Arg - 
Pro-Pro-Glu-Glu-Pro-Ala-Val-His-Ala-Asp-Cys-His-Arg-Ala- 
Ser-Leu-Asn-Ile-Ser-Phe-Gln-Glu-Leu-Gly-Trp-Asp-Arg-Trp- 
Ile-Val-His-Pro-Pro-Ser-Phe-Ile-Phe-His-Tyr-Cys-His-Gly- 
Gly-Cys-Gly-Leu-Pro-Thr-Leu-Pro-Asn-Leu-Pro-Leu-Ser-Val- 
Pro-Gly-Ala-Pro-Pro-Thr-Pro-Val-Gln-Pro-Leu-Leu-Leu-Val- 
Pro-Gly-Ala-Gln-Pro-Cys-Cys-Ala-Ala-Leu-Pro-Gly-Thr-Met- 
Arg-Ser-Leu-Arg-Val-Arg-Thr-Thr-Ser-Asp-Gly-Gly-Tyr-Ser- 
Phe-Lys-Tyr-Glu-Thr-Val-Pro-Asn-Leu-Leu-Thr-Gln-His-Cys- 
Ala-Cys-Ile-OH and is glycosylated. 



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PCT/US86/01S05 



-23- 

9. A protein according to Claim^S wherein 
saia first chain has the formula: H-Ser-Thr-Ala-Pro-Leu- 
Pro-Trp-Pro-Trp-Ser-Pro-Ala-Ala-Leu-Arg-Leu-Leu-Gln-Arg- 
Pro-Pro-Glu-Glu-Pro-Ala-Val-His-Ala-Asp-Cys-His-Arg-Ala- 
5 Ser-Leu-Asn-Ile-Ser-Phe-Gln-Glu-Leu-Gly-Trp-Asp-Arg-Trp- 
Ile-Val-His-Pro-Pro-Ser-Phe-Ile-Phe-His-Tyr-Cys-His-Gly- 
Gly-Cys-Gly-Leu-Pro-Thr-Leu-Pro-Asn-Leu-Pro-Leu-Ser-Val- 
Pro-Gly-Ala-Pro-Pro-Thr-Pro-Val-Gln-Pro-Leu-Leu-Leu-Val- 
Pro-Gly-Ala-Gln-Pro-Cys-Cys-Ala-Ala-Leu-Pro-Gly-Thr-Met- 
10 Ar g - Se r -Leu- Ar g -Val -Ar g -Thr -Thr -Ser -Asp-Gly-Gly-Ty r -Ser - 
Phe-Lys-Tyr-Glu-Thr-Val-Pro-Asn-Leu-Leu-Thr-Gln-His-Cys- 
Ala-Cys-Ile-OH, 

10. A protein according to claim 1 wherein 
said second chain has the formula: 

15 H-Giy-Leu-Glu-Cys-Asp-Gly-Arg-Thr-Asn-Leu-Cys-Cys-Arg-Gln- 
Gln-Phe-Phe-Ile-Asp-Phe-Arg-Leu-Ile-Gly-Trp-Ser-Asp-Trp- 
Ile-Ile-Ala-Pro-Thr-Gly-Tyr-Tyr-Gly-Asn-Tyr-Cys-Glu-Gly- 
Ser-Cys-Pro^Ala-Tyr-Leu-Ala-Gly-Val-Pro-Gly-Ser-Ala-Ser- 
Ser-Phe-His-Thr-Ala-Val-Val-Asn-Gln-Tyr-Arg-Met-Arg-Gly- 

20 Leu-Asn-Pro-Gly-Thr-Val-Asn-Ser-Cys-Cys-Ile-Pro-Thr-Lys- 
Leu-Ser-Thr-Met-Ser-Met-Leu-Tyr-Phe-Asp-Asp-Glu-Tyr-Asn- 
Ile-Val-Lys-Arg-Asp-Val-Pro-Asn-Met-Ile-Val-Glu-Glu-Cys- 
Gly-Cys-Ala-OH 

11. A protein according to Claim 10 wherein 

25 said first chain has the formula: H-Ser-Thr -Ala-Pro-Leu- 
Pro- Trp-Pro-Trp- Ser -Pro- Ala- Ala-Leu- Ar-g -Leu- Leu-Gln-Arg - 
Pro-Pro-Glu-Glu-Pro-Ala-Val-His-Ala-Asp-Cys-His-Arg-Ala- 
Ser-Leu-Asn-Ile-Ser-Phe-Gln-Glu-Leu-Gly-Trp-Asp-Arg-Trp- 
Ile-Val-His-Pro-Pro-Ser-Phe-Ile-Phe-His-Tyr-Cys-His-Gly- 

30 Gly-Cys-Gly-Leu-Pro-Thr-Leu-Pro-Asn-Leu-Pro-Leu-Ser-Val- 
Pro-Gly-Ala-Pro-Pro-Thr-Pro-Val-Gln-Pro-Leu-Leu-Leu-Val- 
Pro-Gly-Ala-Gln-Pro-Cys-Cys-Ala-Ala-Leu-Pro-Gly-Thr -Met- 
Arg-Ser-Leu-Arg-Val-Arg-Thr-Thr-Ser-Asp-Gly-Gly-Tyr-Ser- 
Phe-Lys-Tyr -Glu-Thr -Val-Pr o-Asn-Leu-Leu-Thr -Gin- His -Cys - 

35 Ala-Cys-Ile-OH. 



wo 87/00528 



PCT/US86/01S05 



-24- 

12. A method of obtaining substantially 
homogeneous proteins having inhibin activity from crude 
extracts comprising 

procuring extract material having inhibin 

5 activity, 

exposing said extract to a support medium to 
which heparin moieties are attached under such 
conditions that inhibin proteins adsorb to said heparin 
moieties, and subsequently eluting said inhibin proteins 
10 from said heparin moieties, 

gel filtrating said eluted inhibin proteins and 
selecting fractions having inhibin activity, and 

fractionating said gel filtrated fractions on 
at least one reverse-phase high performance liquid 
15 cnromatography column. 

13. A method according to Claim 12 wherein 
said gel filtrated fractions are fractionated on at 
least two successive reverse-phase high performance 
liquid chromatography columns of different solid and/or 

20 liquid phase conditions. 

14. A method according to Claim 12 wherein at 
least one of said RP-HPLCS is performed on a C3-C5 
column. 

15. A metnod according to Claim 12 wherein 
25 said crude extract is obtained from porcine body 

material. 

16. A method according to Claim 15 wherein 
said body material is porcine follicular fluid. 



wo 87/00528 



PCT/US86/0150S 



-25- 

17. A synthetic protein produced by 
recombinant DNA techniques or the like having two chains 
interconnected by disulfide bonding, said first chain 
having formula: H-Ser-Thr-Ala-Pro-Leu-Pro-Trp-Pro-Trp- 
5 Ser-Pro-Ala-Ala-Leu-Arg-Leu-Leu-Gln-Arg-Pro-Pro-<31u-Glu- 
Pro-Ala-Val-His-Ala-Asp-Cys-His-Arg-Ala-Ser-Leu-Asn-Ile- 
Ser-Phe-Gln-Glu-Leu-Gly-Trp-Asp-Arg-Trp-Ile-Val-His-Pro- 
Pro-Ser-Phe-Ile-Phe-His-Tyr-Cys-His-Gly-Gly-Cys-Gly-Leu- 
Pro-Thr-Leu-Pro-Asn-Leu-Pro-Leu-Ser-Val-Pro-Gly-Ala-Pro- 

10 Pro-Thr-Pro-Val-Gln-Pro-Leu-Leu-Leu-Val-Pro-Gly-Ala-Gln- 
Pro-Cys-Cys-Ala-Ala-Leu-Pro-Gly-Thr-Met-Arg-Ser-Leu-Arg- 
Val-Arg-Thr-Thr-Ser-Asp-Gly-Gly-Tyr-Ser-Phe-Lys-Tyr-Glu- 
Thr-Val-Pro-Asn-Leu-Leu-Thr-Gln-His-Cys-Ala-Cys-Ile-OH, 
and said second chain having either the formula: H-Gly- 

15 Leu-Glu-Cys-Asp-Gly-Lys-Val-Asn-Ile-Cys-Cys-Lys-Lys-Gln- 
Phe-Phe-Val-Ser-Phe-Lys-Asp-Ile-Gly-Trp-Asn-Asp-Trp-Ile- 
Ile-Ala-Pro-Ser-Gly-Tyr-His-Ala-A^n-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- 

20 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, or the formula H-Gly-Leu-Glu-Cys-Asp- 
Gly-Arg-Thr-Asn-Leu-Cys -Cys-Arg-Gln-Gln-Phe-Phe-Ile-Asp- 

2.5 Phe-Arg-Leu-Ile-Gly-Trp-Ser-Asp-Trp-Ile-Ile-Ala-Pro-Thr- 
Gly-Tyr -Tyr -Gly- Asn-Tyr -Cys-Glu-Gly-Ser -Cys -Pr o-Ala-Tyr - 
Leu-Ala-Gly-Val-Pro-Gly-Ser-Ala-Ser-Ser-Phe-His-Thr-Ala- 
Vai-Val-Asn-Gln-Tyr-rArg-Met-Arg-Gly-Leu-Asn-Pro-Gly-Thr- 
Val-Asn-Ser-Cys-Cys-Ile-Pro-Thr-Lys-Leu-Ser-Thr-Met-Ser- 

30 Met-Leu-Tyr-Phe-Asp-Asp-Glu-Tyr-Asn-Ile-Val-Lys-Arg-Asp- 
Val-Pro-Asn-Met-Ile-Val-Glu-Glu-Cys-Gly-Cys-Ala-OH 



wo 87/00528 



PCT/US86/0150S 



-Ze- 
is. A synthetic polypeptide useful as a 
chemical intermediate for the production of a bio- 
logically active dimer, which polypeptide is selected 
from the group consisting of: (a) H-Ser-Thr-Ala-Pro- 
5 Leu-Pro-Trp-Pro-Trp-Ser-Pro-Ala-Ala-Leu-Arg-Leu-Leu-Gln- 
Arg-Pro-Pro-Glu-Glu-Pro-Ala-Val-His-Ala-Asp-Cys-His-Arg- 
Ala-Ser-Leu-Asn-Ile-Ser-Phe-Gln-Glu-Leu-Gly-Trp-Asp-Arg- 
Trp-Ile-Val-His-Pro-Pro-Ser-Phe-Ile-Phe-His-Tyr-Cys-His- 
Gly-Gly-Cys-Gly-Leu-Pro-Thr-Leu-Pro-Asn-Leu-Pro-Leu-Ser- 

10 val-Pro-Gly-Ala-Pro-Pro-Thr-Pro-Val-Gln-Pro-Leu-Leu-Leu- 
Val-Pro-Gly-Ala-Gln-Pro-Cys-Cys-Ala-Ala-Leu-Pro-Gly-Thr- 
Met-Arg-Ser-Leu-Arg-Val-Arg-Thr-Thr-Ser-Asp-Gly-Gly-Tyr- 
Ser-Phe-Lys-Tyr-Glu-Thr-Val-Pro-Asn-Leu-Leu-Thr-Gln-His- 
Cys-Ala-Cys-Ile-OH; (b) H-Gly-Leu-Glu-Cys-Asp-Gly-Arg- 

15 Thr-Asn-Leu-Cys-Cys-Arg-Gln-Gln-Phe-Phe-Ile-Asp-Phe-Arg- 
Leu-Ile-Gly-Trp-Ser-Asp-Trp-Ile-Ile-Ala-Pro-Thr-Gly-Tyr- 
Tyr-Gly-Asn-Tyr-Cys-Glu-Gly-Ser-Cys-Pro-Ala-Tyr-Leu-Ala- 
Gly-Val-Pro-Gly-Ser-Ala-Ser-Ser-Phe-His-Thr-Ala-Val-Val- 
Asn-Gln-Tyr-Arg-Met-Arg-Gly-Leu-Asn-Pro-Gly-Thr-Val-Asn- 

20 Ser-Cys-Cys-Ile-Pro-Thr-Lys-Leu-Ser-Thr-Met-Ser-Met-Leu- 
Tyr-Phe-Asp-Asp-Glu-Tyr-Asn-Ile-Val-Lys-Arg-Asp-Val-Pro- 
Asn-Met-Ile-Val-GluH31u-Cys-Gly-Cys-Ala-0H and (c) H-Gly- 
Leu-Glu-Cys-Asp-Gly-Lys-Val-Asn-Ile-Cys-Cys-Lys-Lys-Gln- 
Phe-Phe-Val-Ser-Phe-Lys-Asp-Ile-Gly-Trp-Asn-Asp-Trp-Ile- 

25 iie-Ala-Pro-Ser-Gly-Tyr-His-Ala-Asn-Tyr-Cys-Glu-Gly-<31u- 
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- 

30 ile-Ile-Lys-Lys-Asp-Ile-Gln-Asn-Met-Ile-Val-Glu-Glu-Cys- 

Gly-Cys-Ser-OH. 



wo 87/00528 



PCT/US86/0150S 



-27- 

19. A polypeptide according to Claim 18 having 
the formula: H-Gly-Leu-Glu-Cys-Asp-Gly-Arg-Thr-Asn-Leu- 
Cys-Cys-Arg-Gln-Gln-Phe-Phe-Ile-Asp-Phe-Arg-Leu-Ile-Gly- 
Trp-Ser-Asp-Trp-Ile-Ile-Ala-Pro-Thr-Gly-Tyr-Tyr-Gly-Asn- 

5 Tyr-Cys-Glu-Gly-Ser-Cys-Pro-Ala-Tyr-Leu-Ala-Gly-Val-Pro- 
Gly-Ser-Ala-Ser-Ser-Phe-His-Thr-Ala-Val-Val-Asn-Gln-Tyr- 
Arg-Met-Arg-Gly-Leu-Asn-Pro-Gly-Thr-Val-Asn-Ser-Cys -Cys- 
Ile-Pro-Thr-Lys-Leu-Ser-Thr-Met-Ser-Met-Leu-Tyr-Phe-Asp- 
Asp-Glu-Tyr-Asn-Ile-Val-Lys-Arg-Asp-Val-Pro-Asn-Met-Ile- 
10 Val-Glu-Glu-Cys-Gly-Cys-Ala-OH. 

20. A polypeptide according to Claim 18 having 
the formula: H-Gly-Leu-Glu-Cys-Asp-Gly-Lys-Val-Asn-Ile- 
Cys-Cys-Lys-Lys-Gln-Phe-Phe-Val-Ser-Phe^Lys -Asp-Ile-Gly- 
Trp-Asn-Asp-Trp-Ile-Ile-Ala-Pro-Ser-Gly-Tyr-His-Ala-Asn- 

15 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- 

20 Ile-Val-Glu-Glu-Cys-Gly-Cys-Ser-OH. 



wo 87/00528 



PCT/US86/01505 



1/5 



9 r 4500 



FIG. la 



8 - 



o 

CM 
< 



1.0 r- 



-i 



I 



I 

I 



a. 



























FIG. lb 



0.5 



o 

00 
CVJ 

< 



3500 h 
3000 
2600 



1500 
1000 
500 
0 



2500 



2000 



1500 l^-g 



25 20 15 10 

FRACTION NUMBER 



1000 jr 



500 




35 30 25 

FRACTION NUMBER 



FIG. Ic 

0.03 



3500 



0.02 



o 

03 
CM 
< 



0.01 



0.00 



500 
0 




S,1*^::;.i>^^B^ 



-J 
2 

o 

I- 

LU 

o 
< 

2« 



30 25 20 15 10 
f RACT10N NUMBER 



10 



wo 87/00528 



PCT/US86/01S05 



FIG. 2a 



2/5 



0.15 r 



o 0.10 - CO 2000 



0.0S 




. 25 20 15 
FRACTION NUMBER 



10 



FIG. 2b 



4000 



0.3 r 



0.2 



o 

CM 

< 



0.1 





imiiiitnimi 



90 
80 
70 



45 40 35 30 25 20 IS 10 

FRACTION NUMBER 



FIG. 2c 



0.75 



0.50 



eg 
< 



0^5 




QliHiiMMHiiimmiinmmimiii n m ilp 

40 35 30 25 20 15 10 5 
FRACTION NUMBER 



wo 87/00528 



PCT/US86/0150S 



3/5 



CO 



00 



I I I 




CD 



t t i 



ro 
O 



CO 

CO 



iri 

CVJ 
CO 



in 




03 



CD 



t t t 



1^ CO 
CO 



to 

CSJ 



to 



wo 87/00528 



PCT/US86/0150S 




(sjM8t7.|uu/6u) HSd 



wo 87/00528 



PCT/US86/01S05 



ai6 r 



FIG. 5a 



0.10 



0.05 



5/5 




JXLLLLLLLL 



11 1 ru inn 



n n 1 1 1 1 i 



3S 



90 
60 
70 

I 

60 I 
50 I 

40 % 
i 

30 
20 
10 



30 25 20 IS 
FRACTION NUMBER 



10 



0.4 



0.3 



FIG. 5b I 



r- 4000 

3500 
3000 
i 2500 



\ 

L \ 



o 0.2 - g 2000 



0.1 - 



0.0 *- 0 - 





90 

SO 
70 
60 
50 
40 
30 
20 
10 



"""""" I X I I 



40 35 30 25 20 IS 10 5 

FRACTION NUMBER 



1.5 



FIG. 50 



1.0 



ru 
< 



0.S 



\ 



\ 



\ 



\ 



\ 



\ 



\ 



\ 



00 It I I I I miHin iiiHiiii Ill 

40 39 30 8S 20 IS 10 5 
fRACDON NUMBER 



80 
70 

I 

50 I 
40 I 
30 ^ 
20 
10 
0 



INTERNATIONAL SEARCH REPORT 

International A pplication No PCT /US 86/01505 

I, CLASSIFICATION OF SUBJECT MATTER (i! several classiHcation symbols apply. Indicate all) a 
Accoidino to International Patent Classification (IPC) or to both National Classirication and IPC 

TNT CL.4 C07K 7/06, 7/08, 7/10, 13/00, 15/00 

TTS PL 124. 325, 326, 327, 328. 344 

n. FIELDS SEARCHED i— f 

^ Minimum Documentation Searched * 

Classincation System I Classification Symbols 

U.S. ' 530/313, 324, 325, 326, 327, 328, 344 

I 

t 

Documentation Searched other than Minimum Documentation 
to the Extent that such Documents are Included In the Fields Searched & 



III. DOCUMENTS CONSIDERED TO BE RELEVANT m 



Category 



I 



Citation of Document, '<> with indication, where appropriate, of the relevant passages 



Relevant to Claim No. is 



X 



US,N, Biochemical & Bjophysjcal Research 1-20 
CoiTtmuhication , PubXisHed 14 JUNE 1985, ^^ol 
129, No. 2, MIYAMnTO et al "Tsolation of 
Porcine Follicular Fluid Jnhibin of 32K 
Daltons," pages 396-403. 

UF,N, archives of Andrology , Published 13 1,12-16 
DECEMBER 198 4, Vol.13, CHT^NNtnG et al 
"Porcine Inhjbin: Initial Fractionation as 
a High Molecular weight Complex," p. 219*423]. 

UP,N, Journal of Chromatography , Published 1,12-^16 
1984, Vol.288, PTwiET? et al "^eversed- 
Phase High-Performance Liouid Chromatograplfy 
Preparative Purification of Synthetic 
Peptides," pp. 303-328. 

US,N, Biology of reproduction , Published ' 
1986, Vol.34, MTLLS et al "The Effects of 
Porcine Follicular Fluid Inhibin on 
Gonadotropin secretion in the Rabbit," 
pp, 336-343. 



* special categories of cited documents: 

"A" document defining the general state of the art which is not 
considered to be of particular relevance 

earlier document but published on or after the international 
filing date 

"L** document which may throw doubts on priority claim(s) or 
which is cited to establish the publication date of another 
citation or other special reason (as specified) 

"O" document referring to an oral disclosure, use, exhibition or 
other means 

"P** document published prior to the international filing date but 
later than the priority date claimed 



"T** later document published after the International filing date 
or priority date and not in conflict with the application but 
cited to understand the principle or theory underlying the 
invention 

''X" document of particular relevance; the claimed invention 
cannot be •considered novel or cannot be considered to 
involve an inventive step 

"Y" document of particular relevance; the claimed invention 
cannot be considered to Involve an inventive step when the 
document is combined with one or more other such docu- 
ments, such combination being obvious to a person skilled 
in the art. 

document member of the same patent family 



IV. CERTIFICATION 



Date of the Actual Completion of the International Search > 
12 AUGUST 1986 


Date of Mailing of this International Search Report * 

2 9 AUG 1986 


International Searching Authority i 

ISA/us 


NATHAN M, NUTTER 



Form PCT/ISA/210 (second sheet) (May 1986} 



International Applicatron No. 



PCT/UP86/01505 



III. DOCUMENTS CONSIDERED TO BE RELEVANT (CONTINUED FROM THE SECOND SHEET) 



Category* | Citation ol Document, »« with indication, where appropriate, of the relevant passages »7 | Relevant to Claim No J 



U<=,P, Chem Abst> 103: 65074s, Published 02 
SEPTEMBER 1985, Vol. 103, No. 9 "Isolation 
From Porcine Follicular Fluid of a Po-tent 
Inhibin-like Biological Activity," 
"RT.VIER et al- 



1-20 



Form PCT/ISA/21 0 (extra sheet) (May 1 986) 



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