Human inhibin

• US 4,737,578 A
• Filed: 04/07/1986
• Issued: 04/12/1988
• Est. Priority Date: 02/10/1986
• Status: Expired due to Term

• Current Assignee: Salk Institute for Biological Studies
• Original Assignee: Salk Institute for Biological Studies
• Inventors: Cerelli, Gail, Mayo, Kelly E., Vale, Wylie W. Jr., Evans, Ronald M., Rosenfeld, Michael G., Rivier, Jean E. F., Spiess, Joachim
• Primary Examiner(s): Phillips, Delbert R.
• Application Number:
  US06/848,924
• Time in Patent Office: 736 Days
• Field of Search: 530/350, 530/324
• US Class Current: 530/350
• 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

Proteins with inhibin activity having a weight of about 32,000 daltons. The 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. The 18K chain is obtained from the human inhibin gene and has the formula: H-Ser-Thr-Pro-Leu-Met-Ser-Trp-Pro-Trp-Ser-Pro-Ser-Ala-Leu-Arg-Leu-Leu-Gln-Arg-Pro-Pro-Glu-Glu-Pro-Ala-Ala-His-Ala-Asn-Cys-His-Arg-Val-Ala-Leu-Asn-Ile-Ser-Phe-Gln-Glu-Leu-Gly-Trp-Glu-Arg-Trp-Ile-Val-Tyr-Pro-Pro-Ser-Phe-R.sub.65 -Phe-His-Tyr-Cys-His-Gly-Gly-Cys-Gly-Leu-His-Ile-Pro-Pro-Asn-Leu-Ser-Leu-Pro-Val-Pro-Gly-Ala-Pro-Pro-Thr-Pro-Ala-Gln-Pro-Tyr-Ser-Leu-Leu-Pro-Gly-Ala-Gln-Pro-Cys-Cys-Ala-Ala-Leu-Pro-Gly-Thr-Met-Arg-Pro-Leu-His-Val-Arg-Thr-Thr-Ser-Asp-Gly-Gly-Tyr-Ser-P e-Lys-Tyr-Glu-Thr-Val-Pro-Asn-Leu-Leu-Thr-Gln-His-Cys-Ala-Cys-Ile-OH, wherein R₆₅ is Ile or Arg. The 18K chain is connected by disulfide bonding to the 14K chain.

Claims

1. A synthetic protein produced using recombinant DNA techniques or the like having two chains interconnected by disulfide bonding, said first chain having formula:

H-Ser-Thr-Pro-Leu-Met-Ser-Trp-Pro-Trp-Ser-Pro-Ser-Ala-Leu-Arg-Leu-Leu-Gln-Arg-Pro-Pro-Glu-Glu-Pro-Ala-Ala-His-Ala-Asn-Cys-His-Arg-Val-Ala-Leu-Asn-Ile-Ser-Phe-Gln-Glu-Leu-Gly-Trp-Glu-Arg-Trp-Ile-Val-Tyr-Pro-Pro-Ser-Phe-R.sub.65 -Phe-His-Tyr-Cys-His-Gly-Gly-Cys-Gly-Leu-His-Ile-Pro-Pro-Asn-Leu-Ser-Leu-Pro-Val-Pro-Gly-Ala-Pro-Pro-Thr-Pro-Ala-Gln-Pro-Tyr-Ser-Leu-Leu-Pro-Gly-Ala-Gln-Pro-Cys-Cys-Ala-Ala-Leu-Pro-Gly-Thr-Met-Arg-Pro-Leu-His-Val-Arg-Thr-Thr-Ser-Asp-Gly-Gly-Tyr-Ser-Phe-Ly -Tyr-Glu-Thr-Val-Pro-Asn-Leu-Leu-Thr-Gln-His-Cys-Ala-Cys-Ile-OH, wherein R₆₅ is Ile or Arg, 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-Ile-A n-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-Val-A n-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.

2. A synthetic peptide according to claim 1 wherein R₆₅ is Ile.

3. A synthetic peptide according to claim 1 wherein R₆₅ is Arg.

4. A synthetic inhibin protein having the sequence:

Ser-Thr-Pro-Leu-Met-Ser-Trp-Pro-Trp-Ser-Pro-Ser-Ala-Leu-Arg-Leu-Leu-Gln-Arg-Pro-Pro-Glu-Glu-Pro-Ala-Ala-His-Ala-Asn-Cys-His-Arg-Val-Ala-Leu-Asn-Ile-Ser-Phe-Gln-Glu-Leu-Gly-Trp-Glu-Arg-Trp-Ile-Val-Tyr-Pro-Pro-Ser-Phe-R₆₅ -Phe-His-Tyr-Cys-His-Gly-Gly-Cys-Gly-Leu-His-Ile-Pro-Pro-Asn-Leu-Ser-Leu-Pro-Val-Pro-Gly-Ala-Pro-Pro-Thr-Pro-Ala-Gln-Pro-Tyr-Ser-Leu-Leu-Pro-Gly-Ala-Gln-Pro-Cys-Cys-Ala-Ala-Leu-Pro-Gly-Thr-Met-Arg-Pro-Leu-His-Val-Arg-Thr-Thr-Ser-Asp-Gly-Gly-Tyr-Ser-Phe-Ly -Tyr-Glu-Thr-Val-Pro-Asn-Leu-Leu-Thr-Gln-His-Cys-Ala-Cys-Ile or the sequence;

Met-Val-Leu-His-Leu-Leu-Leu-Phe-Leu-Leu-Leu-Thr-Pro-Gln-Gly-Gly-His-Ser-Cys-Gln-Gly-Leu-Glu-Leu-Ala-Arg-Glu-Leu-Val-Leu-Ala-Lys-Val-Arg-Ala-Leu-Phe-Leu-Asp-Ala-Leu-Gly-Pro-Pro-Ala-Val-Thr-Arg-Glu-Gly-Gly-Asp-Pro-Gly-Val-Arg-Arg-Leu-Pro-Arg-Arg-His-Ala-Leu Gly-Gly-Phe-Thr-His-Arg-Gly-Ser-Glu-Pro-Glu-Glu-Glu-Glu-Asp-Val-Ser-Gln-Ala-Ile-Leu-Phe-Pro-Ala-Thr-Asp-Ala-Ser-Cys-Glu-Asp-Lys-Ser-Ala-Ala-Arg-Gly-Leu-Ala-Gln-Glu-Ala-Glu-Glu-Gly-Leu-Phe-Arg-Tyr-Met-Phe-Arg-Pro-Ser-Gln-His-Thr-Arg-Ser-Arg-Gln-Val-Thr-Ser Ala-Gln-Leu-Trp-Phe-His-Thr-Gly-Leu-Asp-Arg-Gln-Gly-Thr-Ala-Ala-Ser-Asn-Ser-Ser-Glu-Pro-Leu-Leu-Gly-Leu-Leu-Ala-Leu-Ser-Pro-Gly-Gly-Pro-Val-Ala-Val-Pro-Met-Ser-Leu-Gly-His-Ala-Pro-Pro-His-Trp-Ala-Val-Leu-His-Leu-Ala-Thr-Ser-Ala-Leu-Ser-Leu-Leu-Thr-His-Pro Val-Leu-Val-Leu-Leu-Leu-Arg-Cys-Pro-Leu-Cys-Thr-Cys-Ser-Ala-Arg-Pro-Glu-Ala-Thr-Pro-Phe-Leu-Val-Ala-His-Thr-Arg-Thr-Arg-Pro-Pro-Ser-Gly-Gly-Glu-Arg-Ala-Arg-Arg-Ser-Thr-Pro-Leu-Met-Ser-Trp-Pro-Trp-Ser-Pro-Ser-Ala-Leu-Arg-Leu-Leu-Gln-Arg-Pro-Pro-Glu-Glu-Pro Ala-Ala-His-Ala-Asn-Cys-His-Arg-Val-Ala-Leu-Asn-Ile-Ser-Phe-Gln-Glu-Leu-Gly-Trp-Glu-Arg-Trp-Ile-Val-Tyr-Pro-Pro-Ser-Phe-R₆₅ -Phe-His-Tyr-Cys-His-Gly-Gly-Cys-Gly-Leu-His-Ile-Pro-Pro-Asn-Leu-Ser-Leu-Pro-Val-Pro-Gly-Ala-Pro-Pro-Thr-Pro-Ala-Gln-Pro-Tyr-Ser-Leu-Leu-Pro-Gly-Ala-Gln-Pro-Cys-Cys-Ala-Ala-Leu-Pro-Gly-Thr-Met-Arg-Pro-Leu-His-Val-Arg-Thr-Thr-Ser-Asp-Gly-Gly-Tyr-Ser-Phe-Ly -Tyr-Glu-Thr-Val-Pro-Asn-Leu-Leu-Thr-Gln-His-Cys-Ala-Cys-Ile, wherein R₆₅ is Ile or Arg.

5. A protein according to claim 4 wherein R₆₅ is Ile.

6. A synthetic inhibin protein segment of a protein in accordance with claim 4, which segment is effective to create antibodies which neutralize the FSH-release-inhibiting property of human inhibin.

7. A synthetic segment in accordance with claim 6 wherein said segment constitutes the N-terminal 6-residue sequence Ser-Thr-Pro-Leu-Met-Ser or the 25-residue sequence Ser-Thr-Pro-Leu-Met-Ser-Try-Pro-Trp-Ser-Pro-Ser-Ala-Leu-Arg-Leu-Leu-Gln-Arg-Pro-Pro-Glu-Glu-Pro-Ala of the α

-chain of of human inhibin.