P. pastoris pastoris promoters, and the use thereof to direct expression of proteins in yeast, preferably using a haploid mating strategy

Patent metadata

Field	Value
Publication number	US-9765321-B2
Application number	US-201414323678-A
Country	US
Kind code	B2
Filing date	Jul 3, 2014
Priority date	Oct 10, 2006
Publication date	Sep 19, 2017
Grant date	Sep 19, 2017

Abstract

Novel promoters which are derived from P. pastoris pastoris which are inducible or repressible under specific growth conditions are provided. These promoters are useful for regulating the expression of a desired structural gene, e.g., a mammalian polypeptide. Particularly preferred is the use of these novel promoters to regulate gene expression in polyploidal yeast such as diploidal P. pastoris produced by mating or spheroplast fusion.

First claim

What is claimed is:

1. An isolated nucleic acid sequence containing an enolase (ENOI) promoter derived from the P. pastoris ENOI gene having the nucleic acid sequence contained in SEQ ID NO:22, which when operably linked to a structural gene renders the expression of the gene methanol repressive, and which is operably linked to a heterologous structural gene.

2. The isolated nucleic acid sequence of claim 1 wherein said structural gene encodes a non-yeast protein.

3. The isolated nucleic acid sequence of claim 2 wherein the structural gene is a mammalian gene.

4. The isolated nucleic acid sequence of claim 2 wherein the structural gene encodes an antibody polypeptide.

5. A plasmid containing the isolated nucleic acid of claim 1 .

6. A plasmid containing the isolated nucleic acid of claim 2 .

7. A plasmid containing the isolated nucleic acid of claim 3 .

8. A plasmid containing the isolated nucleic acid of claim 4 .

9. A yeast cell comprising an isolated nucleic acid according to claim 1 , wherein the structural gene is other than ENO1.

10. A yeast cell comprising an isolated nucleic acid according to claim 1 , wherein the structural gene is a mammalian structural gene.

11. A yeast cell comprising an isolated nucleic acid according to claim 1 , wherein the structural gene is a sequence encoding an antibody polypeptide.

12. A yeast cell according to claim 9 which is a P. pastoris, Candida, Saccharomyces, Yarrowia, Kluyveromyces, Hansenula , or Schizosaccharomyces yeast cell.

13. The yeast cell of claim 9 which is a P. pastoris yeast cell.

14. The yeast cell of claim 12 which is polyploid.

15. The yeast cell of claim 13 which is polyploid.

16. A method of using a yeast cell according to claim 9 to produce the protein encoded by said structural gene under the regulatory control of said ENO1 promoter.

17. The method of claim 16 wherein the protein is a mammalian protein.

18. The method of claim 17 wherein the mammalian protein is an antibody, cytokine or growth factor.

Assignees

• Alderbio Holdings Llc
• Alderbio Holdings Llc

Inventors

• Cregg James M
• Tolstorukov Ilya I

Classifications

• Y02P20/52

  using catalysts, e.g. selective catalysts · CPC title

• C12N9/0006

  acting on CH-OH groups as donors (1.1) · CPC title

• C12N15/81

  for yeasts · CPC title

• C12Y402/01011

  Phosphopyruvate hydratase (4.2.1.11), i.e. enolase · CPC title

• C07K16/2809

  against the T-cell receptor (TcR)-CD3 complex · CPC title