Production of heterologous polypeptides in yeast

• US 6,861,237 B2
• Filed: 11/30/2001
• Issued: 03/01/2005
• Est. Priority Date: 11/30/2000
• Status: Expired due to Fees

• Current Assignee: Novo Nordisk A/S
• Original Assignee: Novo Nordisk A/S
• Inventors: Andersen, Asser Sloth, Diers, Ivan
• Primary Examiner(s): Guzo, David
• Application Number:
  US10/002,826
• Publication Number:
  US 20050019853A1
• Time in Patent Office: 1,187 Days
• Field of Search: 435/320.1, 435/69.1, 435/69.4, 435/243, 435/254.1, 435/254.11, 435/254.2, 536/23.1, 536/23.5, 536/23.51, 536/23.7, 536/24.1
• US Class Current: 435/69.4
• CPC Class Codes: C07K 14/395 from Saccharomyces
• CPC Class Codes: C07K 14/62 Insulins
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione

Abstract

A process for producing high amount of proteins or polypeptides in yeast is disclosed. The process makes use of the CIT1 yeast promoter or a functional part or variant thereof. Examples of polypeptides which are expressed in high yields are insulin or insulin analogues or GLP1.

Claims

1. A process for making a heterologous, non-bacterial polypeptide or an intermediate therefore in yeast comprising (i) culturing a yeast strain comprising a polynucleotide sequence encoding the polypeptide or an intermediate therefore under suitable culture conditions for expression of the polypeptide or its intermediate, wherein the polynucleotide sequence encoding the polypeptide or its intermediate is expressed under transcriptional control of a yeast CIT1 promoter or a functional part or variant thereof;

and (ii) isolating the expressed product.

2. A process according to claim 1, wherein the CIT1 promoter consists of all or part of the nucleotide sequence of SEQ ID NO:

1.

3. A process according to claim 1, wherein the CIT1 promoter consists of all or part of the nucleotide sequence from 10 to 722 of SEQ ID NO:

1.

4. A process according to claim 1, wherein the CIT1 promoter consists of all or part of the nucleotide sequence from position 150 to 722 of SEQ ID NO:

1.

5. A process according to claim 1 wherein the promoter consists of all or part of the nucleotide sequence from position 150 to 530 of SEQ ID NO:

1.

6. A process according to claim 1, wherein the expressed product is isolated from the culture medium.

7. A process according to claim 1, wherein the heterologous polypeptide is an insulin precursor.

8. A process according to claim 1, wherein the heterologous polypeptide is GLP-1(7-37).

9. A process according to claim 1, wherein the heterologous polypeptide is GLP-1(7-37)Arg34.

10. A process according to claim 1 being a batch process.

16. A polynucleotide construct according to claim 1 further comprising a leader sequence for secretion of the polypeptide.

11. A polynucleotide construct comprising a polynucleotide sequence encoding a non-bacterial polypeptide or an intermediate therefore operably linked to a DNA sequence encoding a CITI yeast promoter or a functional part or variant thereof, wherein said polypeptide is heterologous with respect to the CIT1 promoter.

12. A polynucleotide construct according to claim 11, wherein the promoter consists of all or part of the nucleotide sequence of SEQ ID NO:

1.

13. A polynucleotide construct according to claim 11, wherein the promoter consists of all or part of the nucleotide sequence from 10 to 722 of SEQ ID NO:

1.

14. A polynucleotide construct according to claim 11, wherein the promoter consists of all or part of the nucleotide sequence from position 150 to 722 of SEQ ID NO:

1.

15. A polynucleotide construct according to claim 11, wherein the promoter consists of all or part of the nucleotide sequence from position 150 to 530 of SEQ ID NO:

1.

17. A yeast cell transformed with a polynucleotide construct according to claim 11.

18. A yeast expression vector comprising in proper reading frame (a) a polynucleotide sequence comprising a CIT1 yeast promoter or a functional part or variant thereof, (b) a polynucleotide sequence encoding a non-bacterial heterologous polypeptide or an intermediate therefore, (c) a suitable leader sequence and, optionally (d) a transcription terminator sequence.

19. A yeast cell transformed with a vector according to claim 18.