Inhibin and method of purifying same

• US 4,740,587 A
• Filed: 10/03/1985
• Issued: 04/26/1988
• Est. Priority Date: 07/18/1985
• Status: Expired due to Term

• Current Assignee: Salk Institute for Biological Studies
• Original Assignee: Salk Institute for Biological Studies
• Inventors: Esch, Fred S., Ying, Shao-Yao, Ling, Nicholas C., Guillemin, Roger C. L.
• Primary Examiner(s): Phillips, Delbert R.
• Assistant Examiner(s): Nutter, Nathan M.
• Application Number:
  US06/784,436
• Time in Patent Office: 936 Days
• Field of Search: 530/313, 530/324, 530/325, 530/326, 530/327, 530/328, 530/344, 530/350, 435/70
• US Class Current: 530/313
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/575 Hormones derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin C07K14/665, e.g. corticotropin C07K14/695
• CPC Class Codes: Y10S 930/26 Containing cys-cys disulfide bridge between nonadjacent cysteine residues

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-performance liquid chromatography. Each isolated molecular 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-Leu-Arg-Leu-Leu-Gln-Arg-Pro-Pro-Glu-Glu-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 14K 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-Ile-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 inhibit basal secretion of FSH, but not of LH, in a rat anterior pituitary monolayer culture system. The half-maximal effective does of one is 450 pg/ml and of the other is 900 pg/ml.

Claims

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 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 beginning with 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 said second chain having an amino-terminal sequence beginning with Gly-Leu-Glu-Cys-Asp-Gly, said protein specifically inhibiting basal secretion of 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.

3. 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-X-Cys-Arg-Gln-Gln-Phe-Phe-Ile-Asp-Phe-Arg-Leu-Ile-Gly-Trp, wherein X is an unknown amino acid residue.

4. A protein according to claim 1 wherein said second chain has the amino-terminal sequence:

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.

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-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- le-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.

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, Ile 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, Ile 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-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- ys-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- eu-Leu-Thr-Gln-His-Cys-Ala-Cys-Ile-OH and is glycosylated.

9. A protein according to claim 5 wherein said 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-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- ys-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- eu-Leu-Thr-Gln-His-Cys-Ala-Cys-Ile-OH.

10. A protein according to claim 1 wherein said second chain has 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-Tyr-Cys-Glu-Gly-Ser-Cys-Pro-Ala-Tyr-Leu-Ala-Gly-Val-Pro-Gly-Ser-Ala-Ser-Ser-Phe-His-Thr-Ala-Val- al-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-Val-Glu-Glu-Cys-Gly-Cys-Ala-OH

11. A protein according to claim 10 wherein said 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-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- ys-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- eu-Leu-Thr-Gln-His-Cys-Ala-Cys-Ile-OH.

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

procuring extract material having inhibin 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 from said heparin moieties,

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

14. 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 liquid phase conditions.

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

16. A method according to claim 12 wherein said RP-HPLC is performed on a C3-C5 column.

17. A method according to claim 12 wherein the liquid phase in said RP-HPLC is a gradient of acetonitrile.

18. A method according to claim 12 wherein said crude extract is obtained from porcine body material.

19. A method according to claim 17 wherein said body material is porcine follicular fluid.

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

20. 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- 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-Pr -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 said second chain having either 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-Tyr-Cys-Glu-Gly-Glu-Cys-Pro-Ser-His-Ile-Ala-Gly-Thr-Ser-Gly-Ser-Ser-Leu-Ser-Phe-His-Ser-Thr-Val- le-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, or 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-Tyr-Cys-Glu-Gly-Ser-Cys-Pro-Ala-Tyr-Leu-Ala-Gly-Val-Pro-Gly-Ser-Ala-Ser-Ser-Phe-His-Thr-Ala-Val- al-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-Val-Glu-Glu-Cys-Gly-Cys-Ala-OH.

21. A synthetic protein useful for the production of synthetic inhibin, which protein is selected from the group consisting of:

(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-Glu-Leu-Gly-Trp-Asp-Arg-Trp-Ile-Val-His-Pro-Pro-Ser-Phe-Ile-Phe-His-Tyr-Cys-His-Gly-Gly- ys-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- eu-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-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- al-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- sn-Ile-Val-Lys-Arg-Asp-Val-Pro-Asn-Met-Ile-Val-Glu-Glu-Cys-Gly-Cys-Ala-OH 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-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-I e-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.