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

• US 5,665,568 A
• Filed: 06/02/1995
• Issued: 09/09/1997
• 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): Jacobson, Dian C.
• Assistant Examiner(s): KEMMERER, ELIZABETH
• Application Number:
  US08/459,850
• Time in Patent Office: 830 Days
• Field of Search: 536/23.1, 536/23.5, 536/23.51, 435/320.1, 435/69.4, 435/69.1, 435/172.3, 435/240.1, 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 a porcine or human inhibin β

_A chain polypeptide wherein the porcine inhibin β

_A chain polypeptide has at least the sequence of residues at positions 309-424 of SEQ ID NO;

31, or is a hormonally active variant thereof wherein the variant differs from the porcine inhibin β

_A chain polypeptide of SEQ ID NO;

31 by deletion of the lysine at position 321 or 322, or by substitution of the lysine residue at position 322 with a histidine or serine, or by substitution of the lysine residue at position 315 with an arginine and the valine residue at position 316 with the threonine, or by deletion of the cysteine residue at position 388 or 389, or by substitution of the arginine residue at position 395 with a glutamine, or by substitution of the lysine residue at position 411 with a glutamine, and wherein the human inhibin β

_A chain polypeptide has at least the sequence of residues at positions 311-426 of SEQ ID NO;

41, or is a hormonally active variant thereof wherein the variant differs from the human inhibin β

_A chain polypeptide of SEQ ID NO;

41 by deletion or substitution of an amino acid residue within positions 321-325, 392-404, or 415-426, or by insertion of an amino acid residue adjacent to a residue within said positions, or by substitution of the phenylalanine residue at position 330 with an isoleucine or leucine, or by substitution of tryptophan residue at position 335 with a tyrosine or phenylalanine, or by substitution of the tryptophan residue at position 338 with a tyrosine or phenylalanine, or by substitution of the isoleucine at position 339 with a phenylalanine or valine, or by substitution of the tyrosine residue at position 345 with a tryptophan or threonine, or by substitution of the histidine residue at position 346 with a lysine, or by substitution of the alanine residue at position 347 with a serine, or by substitution of the asparagine residue at position 348 with a glutamine, tyrosine, or histidine, or by substitution of the tyrosine residue at position 349 with a threonine or asparagine, or by substitution of the phenylalanine residue at position 368 with a tyrosine, or by substitution of the histidine residue at position 381 with an asparagine or lysine, or by substitution of the phenylalanine residue at position 384 with a tyrosine.

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

3. The DNA of claim 1 wherein the polypeptide has the amino acid sequence of SEQ ID NO:

31, SEQ ID NO;

41, residues 309-424 of SEQ ID NO;

31, residues 311-426 of SEQ ID NO;

41, or residues 29-426 of SEQ ID NO;

41.

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

5. The DNA of claim 1 wherein the human inhibin β

_A chain polypeptide is a variant wherein the variant differs from the human inhibin β

_A chain polypeptide of SEQ ID NO;

41 by deletion or substitution of an amino acid residue within residues 321-325, 392-404, and 415-426 of SEQ ID NO;

41, with the proviso that the proline, cysteine, and glycine residues within residues 321-325, 392-404, and 415-426 of SEQ ID NO;

41 are not deleted or substituted.

6. The DNA of claim 1 wherein the human inhibin β

_A chain polypeptide is a variant wherein the variant differs from the human inhibin β

_A chain polypeptide of SEQ ID NO;

41 by substitution of the glutamine residue at position 297 with an asparagine or lysine, or by substitution of the valine residue at position 364 with a phenylalanine or leucine, or by substitution of the tyrosine residue at position 375 with a threonine, or by substitution of the tyrosine residue at position 376 with a tryptophan, or by substitution of the asparagine residue at position 389 with a glutamine, histidine, or lysine, or by substitution of the isoleucine residue at position 391 with a leucine or threonine, or by substitution of the methionine residue at position 390 with a leucine or serine, or by substitution of the valine residue at position 392 with a phenylalanine, glutamic acid, threonine, or isoleucine.

7. A replicable vector comprising DNA encoding a porcine or human inhibin β

_A chain polypeptide wherein the porcine inhibin β

_A chain polypeptide has at least the sequence of residues at positions 309-424 of SEQ ID NO;

31, or is a hormonally active variant thereof wherein the variant differs from the porcine inhibin β

_A chain polypeptide of SEQ ID NO;

31 by deletion of the lysine at position 321 or 322, or by substitution of the lysine residue at position 322 with a histidine or serine, or by substitution of the lysine residue at position 315 with an arginine and the valine residue at position 316 with a threonine, or by deletion of the cysteine residue at position 388 or 389, or by substitution of the arginine residue at position 395 with a glutamine, or by substitution of the lysine residue at position 411 with a glutamine, and wherein the human inhibin β

_A chain polypeptide has at least the sequence of residues at positions 311-426 of SEQ ID NO;

41, or is a hormonally active variant thereof wherein the variant differs from the human inhibin β

_A chain polypeptide of SEQ ID NO;

41 by deletion or substitution of an amino acid residue within positions 321-325, 392-404, or 415-426, or by insertion of an amino acid residue adjacent to a residue within said positions, or by substitution of the phenylalanine residue at position 330 with an isoleucine or leucine, or by substitution of the tryptophan residue at position 335 with a tyrosine or phenylalanine, or by substitution of the tryptophan residue at position 338 with a tyrosine or phenylalanine, or by substitution of the isoleucine at position 339 with a phenylalanine or valine, or by substitution of the tyrosine residue at position 345 with a tryptophan or threonine, or by substitution of the histidine residue at position 346 with a lysine, or by substitution of the alanine residue at position 347 with a serine, or by substitution of the asparagine residue at position 348 with a glutamine, tyrosine, or histidine, or by substitution of the tyrosine residue at position 349 with a threonine or asparagine, or by substitution of the phenylalanine residue at position 368 with a tyrosine, or by substitution of the histidine residue at position 381 with an asparagine or lysine, or by substitution of the phenylalanine residue at position 384 with a tyrosine.

8. The vector of claim 7 wherein the polypeptide has the amino acid sequence of SEQ ID NO:

31, SEQ ID NO;

41, residues 309-424 of SEQ ID NO;

31, residues 311-426 of SEQ ID NO;

41, or residues 29-426 of SEQ ID NO;

41.

9. The vector of claim 8 comprising a viral promoter operably linked to the DNA encoding the polypeptide.

10. The vector of claim 7 wherein the human inhibin β

_A chain polypeptide is a variant wherein the variant differs from the human inhibin β

_A chain polypeptide of SEQ ID NO;

41 by deletion or substitution of an amino acid residue within residues 321-325, 392-404, and 415-426 of SEQ ID NO;

41, with the proviso that the proline, cysteine, and glycine residues within residues 321-325, 392-404, and 415-426 of SEQ ID NO;

41 are not deleted or substituted.

11. The vector of claim 7 wherein the human inhibin β

_A chain polypeptide is a variant wherein the variant differs from the human inhibin β

_A chain polypeptide of SEQ ID NO;

41 by substitution of the glutamine residue at position 297 with an asparagine or lysine, or by substitution of the valine residue at position 364 with a phenylalanine or leucine, or by substitution of the tyrosine residue at position 375 with a threonine, or by substitution of the tyrosine residue at position 376 with a tryptophan, or by substitution of the asparagine residue at position 389 with a glutamine, histidine, or lysine, or by substitution of the isoleucine residue at position 391 with a leucine or threonine, or by substitution of the methionine residue at position 390 with a leucine or serine, or by substitution of the valine residue at position 392 with a phenylalanine, glutamic acid, threonine, or isoleucine.

12. A host cell comprising a replicable vector comprising DNA encoding a porcine or human inhibin β

_A chain polypeptide wherein the porcine inhibin β

_A chain polypeptide has at least the sequence of residues at positions 309-424 of SEQ ID NO;

31, or is a hormonally active variant thereof wherein the variant differs from the porcine inhibin β

_A chain polypeptide of SEQ ID NO;

31 by deletion of the lysine at position 321 or 322, or by substitution of the lysine residue at position 322 with a histidine or serine, or by substitution of the lysine residue at position 315 with an arginine and the valine residue at position 316 with a threonine, or by deletion of the cysteine residue at position 388 or 389, or by substitution of the arginine residue at position 395 with a glutamine, or by substitution of the lysine residue at position 411 with a glutamine, and wherein the human inhibin β

_A chain polypeptide has at least the sequence of residues at positions 311-426 of SEQ ID NO;

41, or is a hormonally active variant thereof wherein the variant differs from the human inhibin β

_A chain polypeptide of SEQ ID NO;

41 by deletion or substitution of an amino acid residue within positions 321-325, 392-404, or 415-426, or by insertion of an amino acid residue adjacent to a residue within said positions, or by substitution of the phenylalanine residue at position 330 with an isoleucine or leucine, or by substitution of the tryptophan residue at position 335 with a tyrosine or phenylalanine, or by substitution of the tryptophan residue at position 338 with a tyrosine or phenylalanine, or by substitution of the isoleucine at position 339 with a phenylalanine or valine, or by substitution of the tyrosine residue at position 345 with a tryptophan or threonine, or by substitution of the histidine residue at position 346 with a lysine, or by substitution of the alanine residue at position 347 with a serine, or by substitution of the asparagine residue at position 348 with a glutamine, tyrosine, or histidine, or by substitution of the tyrosine residue at position 349 with a threonine or asparagine, or by substitution of the phenylalanine residue at position 368 with a tyrosine, or by substitution of the histidine residue at position 381 with an asparagine or lysine, or by substitution of the phenylalanine residue at position 384 with a tyrosine.

13. The cell of claim 12 that is a eukaryotic cell.

14. The cell of claim 12 wherein the polypeptide has the amino acid sequence of SEQ ID NO:

31, SEQ ID NO;

41, residues 309-424 of SEQ ID NO;

31, residues 311-426 of SEQ ID NO;

41, or residues 29-426 of SEQ ID NO;

41.

15. The cell of claim 12 wherein the human inhibin β

_A chain polypeptide is a variant wherein the variant differs from the human inhibin β

_A chain polypeptide of SEQ ID NO;

41 by deletion or substitution of an amino acid residue within residues 321-325, 392-404, and 415-426 of SEQ ID NO;

41, with the proviso that the proline, cysteine, and glycine residues within residues 321-325, 392-404, and 415-426 of SEQ ID NO;

41 are not deleted or substituted.

16. The cell of claim 12 wherein the human inhibin β

_A chain polypeptide is a variant the variant differs from the human inhibin β

_A chain polypeptide of SEQ ID NO;

41 by substitution of the glutamine residue at position 297 with an asparagine or lysine, or by substitution of the valine residue at position 364 with a phenylalanine or leucine, or by substitution of the tyrosine residue at position 375 with a threonine, or by substitution of the tyrosine residue at position 376 with a tyrptophan, or by substitution of the asparagine residue at position 389 with a glutamine, histidine, or lysine, or by substitution of the isoleucine residue at position 391 with a leucine or threonine, or by substitution of the methionine residue at position 390 with a leucine or serine, or by substitution of the valine residue at position 392 with a phenylalanine, glutamic acid, threonine, or isoleucine.

17. A method for producing a porcine or human inhibin β

_A chain polypeptide wherein the porcine inhibin β

_A chain polypeptide has at least the sequence of residues at positions 309-424 of SEQ ID NO;

31, or is a hormonally active variant thereof wherein the variant differs from the porcine inhibin β

_A chain polypeptide of SEQ ID NO;

31 by deletion of the lysine at position 321 or 322, or by substitution of the lysine residue at position 322 with a histidine or serine, or by substitution of the lysine residue at position 315 with an arginine and the valine residue at position 316 with a threonine, or by deletion of the cysteine residue at position 388 or 389, or by substitution of the arginine residue at position 395 with a glutamine, or by substitution of the lysine residue at position 411 with a glutamine, and wherein the human inhibin β

_A chain polypeptide has at least the sequence of residues at positions 311-426 of SEQ ID NO;

41, or is a hormonally active variant thereof wherein the variant differs from the human inhibin β

_A chain polypeptide of SEQ ID NO;

41 by deletion or substitution of an amino acid residue within positions 321-325, 392-404, or 415-426, or by insertion of an amino acid residue adjacent to a residue within said positions, or by substitution of the phenylalanine residue at position 330 with an isoleucine or leucine, or by substitution of the tryptophan residue at position 335 with a tyrosine or phenylalanine, or by substitution of the tryptophan residue at position 338 with a tyrosine or phenylalanine, or by substitution of the isoleucine at position 339 with a phenylalanine or valine, or by substitution of the tyrosine residue at position 345 with a tryptophan or threonine, or by substitution of the histidine residue at position 346 with a lysine, or by substitution of the alanine residue at position 347 with a serine, or by substitution of the asparagine residue at position 348 with a glutamine, tyrosine, or histidine, or by substitution of the tyrosine residue at position 349 with a threonine or asparagine, or by substitution of the phenylalanine residue at position 368 with a tyrosine, or by substitution of the histidine residue at position 381 with an asparagine or lysine, or by substitution of the phenylalanine residue at position 384 with a tyrosine, which method comprises;

(a) constructing a vector that comprises a nucleic acid encoding said polypeptide,

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

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

18. The method of claim 17 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.

19. The method of claim 18 wherein the promoter is a viral promoter.

20. The method of claim 19 wherein the promoter is an SV40 promoter.

21. The method of claim 17 wherein the cell is a cell of mammalian origin.

22. The method of claim 17 wherein the cell is a prokaryote.

23. The method of claim 17 wherein the polypeptide has the amino acid sequence of SEQ ID NO:

31, SEQ ID NO;

41, residues 309-424 of SEQ ID NO;

31, residues 311-426 of SEQ ID NO;

41, or residues 29-426 of SEQ ID NO;

41.

24. The method of claim 17 wherein the human inhibin β

_A chain polypeptide is a variant wherein the variant differs from the human inhibin β

_A chain polypeptide of SEQ ID NO;

41 by deletion or substitution of an amino acid residue within residues 321-325, 392-404, and 415-426 of SEQ ID NO;

41, with the proviso that the proline, cysteine, and glycine residues within residues 321-325, 392-404 and 415-426 of SEQ ID NO;

41 are not deleted or substituted.

25. The method of claim 17 wherein the human inhibin β

_A chain polypeptide is a variant wherein the variant differs from the human inhibin β

_A chain polypeptide of SEQ ID NO;

41 by substitution of the glutamine residue at position 297 with an asparagine or lysine, or by substitution of the valine residue at position 364 with a phenylalanine or leucine, or by substitution of the tyrosine residue at position 375 with a threonine, or by substitution of the tyrosine residue at position 376 with a tryptophan, or by substitution of the asparagine residue at position 389 with a glutamine, histidine, or lysine, or by substitution of the isoleucine residue at position 391 with a leucine or threonine, or by substitution of the methionine residue at position 390 with a leucine or serine, or by substitution of the valine residue at position 392 with a phenylalanine, glutamic acid, threonine, or isoleucine.

26. A method for producing a porcine or human inhibin β

_A chain polypeptide wherein the porcine inhibin β

_A chain polypeptide has at least the sequence of residues at positions 309-424 of SEQ ID NO;

31, or is a hormonally active variant thereof wherein the variant differs from the porcine inhibin β

_A chain polypeptide of SEQ ID NO;

31 by deletion of the lysine at position 321 or 322, or by substitution of the lysine residue at position 322 with a histidine or serine, or by substitution of the lysine residue at position 315 with an arginine and the valine residue at position 316 with a threonine, or by deletion of the cysteine residue at position 388 or 389, or by substitution of the arginine residue at position 395 with a glutamine, or by substitution of the lysine residue at position 411 with a glutamine, and wherein the human inhibin β

_A chain polypeptide has at least the sequence of residues at positions 311-426 of SEQ ID NO;

41, or is a hormonally active variant thereof wherein the variant differs from the human inhibin β

_A chain polypeptide of SEQ ID NO;

41 by deletion or substitution of an amino acid residue within positions 321-325, 392-404, or 415-426, or by insertion of an amino acid residue adjacent to a residue within said positions, or by substitution of the phenylalanine residue at position 330 with an isoleucine or leucine, or by substitution of the tryptophan residue at position 335 with a tyrosine or phenylalanine, or by substitution of the tryptophan residue at position 338 with a tyrosine or phenylalanine, or by substitution of the isoleucine at position 339 with a phenylalanine or valine, or by substitution of the tyrosine residue at position 345 with a tryptophan or threonine, or by substitution of the histidine residue at position 346 with a lysine, or by substitution of the alanine residue at position 347 with a serine, or by substitution of the asparagine residue at position 348 with a glutamine, tyrosine, or histidine, or by substitution of the tyrosine residue at position 349 with a threonine or asparagine, or by substitution of the phenylalanine residue at position 368 with a tyrosine, or by substitution of the histidine residue at position 381 with an asparagine or lysine, or by substitution of the phenylalanine residue at position 384 with a tyrosine, which method comprises culturing a host cell comprising nucleic acid encoding said polypeptide under conditions appropriate for producing said polypeptide.

27. The method of claim 26 further comprising the step of recovering the polypeptide from the culture.

28. The method of claim 26 wherein the human inhibin β

_A chain polypeptide is a variant wherein the variant differs from the human inhibin β

_A chain polypeptide of SEQ ID NO;

41 by deletion or substitution of an amino acid residue within residues 321-325, 392-404, and 415-426 of SEQ ID NO;

41, with the proviso that the proline, cysteine, and glycine residues within residues 321-325, 392-404, and 415-426 of SEQ ID NO;

41 are not deleted or substituted.

29. The method of claim 26 wherein the human inhibin β

_A chain polypeptide is a variant wherein the variant differs from the human inhibin β

_A chain polypeptide of SEQ ID NO;

41 by substitution of the glutamine residue at position 297 with an asparagine or lysine, or by substitution of the valine residue at position 364 with a phenylalanine or leucine, or by substitution or the tyrosine residue at position 375 with a threonine, or by substitution of the tyrosine residue at position 376 with a tryptophan, or by substitution of the asparagine residue at position 389 with a glutamine, histidine, or lysine, or by substitution of the isoleucine residue at position 391 with a leucine or threonine, or by substitution of the methionine residue at position 390 with a leucine or serine, or by substitution of the valine residue at position 392 with a phenylalanine, glutamic acid, threonine, or isoleucine.

30. The method of claim 26 wherein the polypeptide has the amino acid sequence of SEQ ID NO:

31, SEQ ID NO;

41, residues 309-424 of SEQ ID NO;

31, residues 311-426 of SEQ ID NO;

41, or residues 29-426 of SEQ ID NO;

41.