Nucleic acid encoding the mature .alpha. chain of inhibin and method for synthesizing polypeptides using such nucleic acid

• US 5,525,488 A
• Filed: 02/17/1994
• Issued: 06/11/1996
• Est. Priority Date: 10/03/1985
• Status: Expired due to Term

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: Seeburg, Peter H., Mason, Anthony J.
• Primary Examiner(s): Walsh, Stephen G.
• Assistant Examiner(s): Fitzgerald, David L.
• Application Number:
  US08/197,792
• Time in Patent Office: 845 Days
• Field of Search: 536/23.5, 435/240.2, 435/320.1, 435/69.4, 435/69.5, 435/254.11, 435/252.3
• US Class Current: 435/69.4
• 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

Abstract

DNA encoding the prepro inhibin α and β chains has been isolated. This DNA is ligated into expression vectors and used to transform host cells for the preparation of inhibin or activin. Also provided are prohormone domains and other inhibin α or β chain derivatives having therapeutic or diagnostic interest. The compositions provided herein are useful in the manipulation of fertility in animals.

Claims

1. Non-chromosomal DNA encoding an inhibin α

chain polypeptide selected from the group consisting of

the porcine inhibin α

chain having the sequence shown as residues 231-364 of SEQ ID NO;

29,

a variant inhibin α

chain having a sequence identical to residues 231-364 of SEQ ID NO;

29 except for the replacement of Asn²⁶⁶ by Gln,

the human inhibin α

chain having the sequence shown as residues 218-351 of SEQ ID NO;

39,

a variant inhibin α

chain having a sequence identical to residues 218-351 of SEQ ID NO;

39 except for the replacement of Asn²³⁷ by Gln, and

a mature mammalian inhibin α

chain polypeptide,

wherein DNA encoding said mammalian polypeptide has the sequence of DNA which is specifically identified on a Southern blot of an ovarian cDNA library using any 64-nucleotide probe within residues 762-1166 of SEQ ID NO;

30 or within residues 654-1058 of SEQ ID NO;

40,

and wherein said mammalian inhibin α

chain polypeptide is capable, not by itself but when combined with a mature mammalian inhibin β

chain polypeptide, of inhibiting the basal release of follicle stimulating hormone, but not of luteinizing hormone, from anterior pituitary cells.

2. The DNA of claim 1 that is free of intervening untranslated sequences.

3. The DNA of claim 1 wherein the polypeptide has an amino acid sequence shown as residues 231-364 of SEQ ID NO:

29 or shown as residues 218-351 of SEQ ID NO;

39.

4. The DNA of claim 1 that is labeled with a detectable moiety.

5. A replicable vector comprising DNA encoding an inhibin α

chain polypeptide selected from the group consisting of

the porcine inhibin α

chain having the sequence shown as residues 231-364 of SEQ ID NO;

29,

a variant inhibin α

chain having a sequence identical to residues 231-364 of SEQ ID NO;

29 except for the replacement of Asn²⁶⁶ by Gln,

the human inhibin α

chain having the sequence shown as residues 218-351 of SEQ ID NO;

39,

a variant inhibin α

chain having a sequence identical to residues 218-351 of SEQ ID NO;

39 except for the replacement of Asn²³⁷ by Gln, and

a mature mammalian inhibin α

chain polypeptide,

wherein DNA encoding said mammalian polypeptide has the sequence of DNA which is specifically identified on a Southern blot of an ovarian cDNA library using any 65-nucleotide probe within residues 762-1166 of SEQ ID NO;

30 or within residues 654-1058 of SEQ ID NO;

40,

and wherein said mammalian inhibin α

chain polypeptide is capable, not by itself but when combined with a mature mammalian inhibin β

chain polypeptide, of inhibiting the basal release of follicle stimulating hormone, but not of luteinizing hormone, from anterior pituitary cells.

6. The vector of claim 5 wherein the polypeptide has an amino acid sequence shown as residues 231-364 of SEQ ID NO:

29 or shown as residues 218-351 of SEQ ID NO;

39.

7. The vector of claim 6 comprising a viral promoter operably linked to the DNA encoding the polypeptide.

8. A host cell transformed with a replicable vector comprising DNA encoding an inhibin α

chain polypeptide selected from the group consisting of

the porcine inhibin α

chain having the sequence shown as residues 231-364 of SEQ ID NO;

29,

a variant inhibin α

chain having a sequence identical to residues 231-364 of SEQ ID NO;

29 except for the replacement of Asn²⁶⁶ by Gln,

the human inhibin α

chain having the sequence shown as residues 218-351 of SEQ ID NO;

39,

a variant inhibin α

chain having a sequence identical to residues 218-351 of SEQ ID NO;

39 except for the replacement of Asn²³⁷ by Gln, and

a mature mammalian inhibin α

chain polypeptide,

wherein DNA encoding said mammalian polypeptide has the sequence of DNA which is specifically identified on a Southern blot of an ovarian cDNA library using any 64-nucleotide probe within residues 762-1166 of SEQ ID NO;

30 or within residues 654-1058 of SEQ ID NO;

40,

and wherein said mammalian inhibin α

chain polypeptide is capable, not by itself but when combined with a mature mammalian inhibin β

chain polypeptide, of inhibiting the basal release of follicle stimulating hormone, but not of luteinizing hormone, from anterior pituitary cells.

9. The cell of claim 8 that is a eukaryotic cell.

10. The cell of claim 8 wherein the polypeptide has an amino acid sequence shown as residues 231-364 of SEQ ID NO:

29 or shown as residues 218-351 of SEQ ID NO;

39.

11. A method for expressing a mature inhibin α

chain polypeptide, comprising the steps of

(a) constructing a vector that comprises a nucleic acid selected from the group consisting of

DNA encoding the porcine inhibin α

chain having the sequence shown as residues 231-364 of SEQ ID NO;

29,

DNA encoding a variant inhibin α

chain having a sequence identical to residues 231-364 of SEQ ID NO;

29 except for the replacement of Asn²⁶⁶ by Gln,

DNA encoding the human inhibin α

chain having the sequence shown as residues 218-351 of SEQ ID NO;

39,

DNA encoding a variant inhibin α

chain having a sequence identical to residues 218-351 of SEQ ID NO;

39 except for the replacement of Asn²³⁷ by Gln, and

DNA encoding a mature mammalian inhibin α

chain polypeptide,

wherein DNA encoding said mammalian polypeptide has the sequence of DNA which is specifically identified on a Southern blot of an ovarian cDNA library using any 64-nucleotide probe within residues 762-1166 of SEQ ID NO;

30 or within residues 654-1058 of SEQ ID NO;

40,

and wherein said mammalian inhibin α

chain polypeptide is capable, not by itself but when combined with a mature mammalian inhibin β

chain polypeptide, of inhibiting the basal release of follicle stimulating hormone, but not of luteinizing hormone, from anterior pituitary cells;

(b) transforming a host cell with the vector; and

(c) culturing the transformed cell under conditions appropriate for the expression of said polypeptide.

12. The method of claim 11 wherein the nucleic acid is operably linked to a promoter recognized by the host cell and comprising further the step of recovering the polypeptide from the culture medium.

13. The method of claim 12 wherein the promoter is a viral promoter.

14. The method of claim 13 wherein the promoter is an SV40 promoter.

15. The method of claim 11 wherein the cell is of mammalian origin.

16. The method of claim 11 wherein the cell is a prokaryote.

17. The method of claim 16 wherein the polypeptide has an amino acid sequence shown as residues 231-364 of SEQ ID NO:

29 or shown as residues 218-351 of SEQ ID NO;

39.