Method for the production of polypeptides

• US 6,110,703 A
• Filed: 07/07/1997
• Issued: 08/29/2000
• Est. Priority Date: 07/05/1996
• Status: Expired due to Term

• Current Assignee: Novo Nordisk A/S
• Original Assignee: Novo Nordisk A/S
• Inventors: Brandt, Jakob, Vad, Knud, Egel-Mitani, Michi
• Primary Examiner(s): Schwartzman, Robert A.
• Assistant Examiner(s): SANDALS, WILLIAM O
• Application Number:
  US08/888,381
• Time in Patent Office: 1,149 Days
• Field of Search: 435/69.1, 435/69.4, 435/255.1, 435/255.2, 435/471, 536/23.1
• US Class Current: 435/69.1
• CPC Class Codes: C12N 15/81 for yeasts

Abstract

The present invention relates to a novel method for the production of short chain polypeptides, including polypeptides having up to 3 disulfide bonds and/or structures rich in basic amino acid residues, and open structured short chain polypeptides, e.g. glucagon, glucagon like peptides and their functional analogues, in genetically modified yeast cells, said genetically modified yeast cells, and a method for the preparation of said yeast cells.

Claims

1. A method for the production of a heterologous short chain polypeptide in yeast, comprising (a) culturing a yeast having reduced activity of Yap 3 protease, said yeast being transformed with a hybrid vector comprising a yeast promoter operably linked to a DNA sequence coding for a polypeptide having up to 55 amino acids and having from 0 to 3 disulfide bonds in the structure, and (b) isolating said polypeptide, wherein said yeast produces said short-chain polypeptide in a yield at least about 2-fold higher than a yeast transformed with said hybrid vector having a wild-type level of Yap3 protease activity.

2. A method according to claim 1, wherein the yeast lacks Yap3 protease activity.

3. A method according to claim 1, wherein the yeast is a diploid yeast.

4. A method according to claim 1 wherein the yeast additionally has reduced protease activity selected from the group of proteases coded for by BAR1, STE13, PRA, PRB, KEX1, PRC, CPS, and the YAP3 homologues MKC7, YAP3-link (coded by GenBank acc. No. X89514:

pos.25352-26878), YIV9 (Swiss Prot acc. No. P40583), and aspartyl protease (IV) (coded by GenBank acc. No. U28372;

pos. 326-2116) genes.

5. A method according to claim 1 wherein the yeast additionally has reduced protease activity selected from the group of proteases coded for by STE13, PRA, PRB, KEX1, and PRC genes.

6. A method according to claim 1, wherein the yeast additionally has reduced protease activity of the aparatyl protease encoded by the yeast open reading frame YGL259W or selected from the group of serine proteases coded for by the KEX2 gene and the yeast open reading frame YCR045C and YOR003W.

7. A method according to claim 1 wherein the yeast additionally has the pep4-3 mutation.

8. A method according to claim 1, wherein the hybrid vector comprises a yeast promoter operably linked to a first DNA sequence encoding a leader sequence being either a pre-sequence (signal) or a prepro-sequence linked in the proper reading frame to a second DNA sequence coding for a polypeptide having from 10-50 amino acids the and having no more than one disulfide bond in the structure, and a DNA sequence containing yeast transcription termination signals.

9. A method according to claim 1 wherein the polypeptide is selected from the group consisting of GRPP (glicentine related polypeptide), glucagon, GLP-1 (glucagon like peptide 1), and GLP-2 (glucagon like peptide 2), GLP-1(7-36), GLP-1(7-35)R36A, GLP-2 F22Y, GLP-2 A19T+34Y, GLP-2 A2G, GLP-2 A19T, and other glucagon analogues having from 1 to 3 amino acid changes, additions and/or deletions.

10. A method according to claim 1, wherein the DNA sequence encoding a polypeptide is selected from the group of DNA sequences encoding CRF and a truncated and/or N-terminally extended form of CART, preferably.

11. A method according to claim 1, wherein the yeast is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces kluyveri, Schizosaccharomyces pombe, Kluyveromyces lactis, Hansenula polymorpha, Pichia pastoris, Pichia methanolica, Pichia kluyveri, Yarrowia lipolytica, Candida sp., Candida utilis, Candida cacaoi, Geotrichum sp., and Geotrichum fermentans.

12. A method according to claim 1, wherein the yeast is S. cerevisiae.

13. A method according to claim 1, wherein the hybrid vector comprises a yeast promoter operably linked to a first DNA sequence encoding a leader sequence being either a pre-sequence (signal) or a prepro-sequence linked in the proper reading frame to a second DNA sequence coding for a polypeptide having up to 55 amino acids, and having from 0 to 3 disulfide bonds in the structure, and a DNA sequence containing yeast transcription signals.

14. A method according to claim 13, wherein the yeast promoter is selected from the group consisting of the MFα

1 promoter, CYC1 promoter, galactose inducible promoters, glycolytic enzyme promoters including TPI and PGK promoters, TRP1 promoter, CUP1 promoter, PHO5 promoter, ADH1 promoter, and HSP promoters.

15. A method according to claim 13, wherein said presequence is selected from the group consisting of:

the signal peptide of mouse α

-amylase, S. cerevisiae MFα

1, BAR1, YAP3 HSP150; and

S. kluyveri MFα

signal peptide; and

said preprosequence is selected from the group consisting of the prepro-sequence of S. cerevisiae MFα

1, YAP3, PRC, HSP150 and S. kluyveri MFα

.

16. A method for the production of a heterologous open structured short chain polypeptide in yeast, comprising (a) culturing a yeast having reduced activity of Yap3 protease, said yeast being transformed with a hybrid vector comprising a yeast promoter operably linked to a DNA sequence coding for a polypeptide having from 20-30 amino acids in the polypeptide chain, and having no more than one disulfide bonds in the structure, and (b) isolating said polypeptide, wherein said yeast produces said short-chain polypeptide in a yield at least about 2-fold higher than a yeast transformed with said hybrid vector having a wild-type level of Yap3 protease activity.

17. A culture of yeast cells containing a DNA sequence encoding a heterologous polypeptide having up to 55 amino acids in the polypeptide chain, and having from 0 to 3 disulfide bonds in the structure, and a second DNA sequense encoding a leader sequence causing said polypeptide to be secreted from the yeast, wherein said culture of yeast cells has reduced Yap3p activity and wherein said yeast produces said polypeptide in a yield at least about 2-fold higher than a yeast containing said DNA sequence and having a wild-type level of Yap3 protease activity.

18. A culture of yeast cells according to claim 17 which is transformed with a hybrid vector comprising said DNA sequence and said second DNA sequence.

19. A method according to claim 18 wherein the yeast is S. cerevisiae.