Alcohol oxidase 1 regulatory nucleotide sequences for heterologous gene expression in yeast

• US 6,699,691 B2
• Filed: 04/05/2002
• Issued: 03/02/2004
• Est. Priority Date: 04/05/2001
• Status: Active Grant

• Current Assignee: Board of Regents of the University of Nebraska
• Original Assignee: Board of Regents of the University of Nebraska
• Inventors: Inan, Mehmet, Benson, Andrew K., Meagher, Michael M.
• Primary Examiner(s): MCKELVEY, TERRY ALAN
• Application Number:
  US10/116,993
• Publication Number:
  US 20030044947A1
• Time in Patent Office: 697 Days
• Field of Search: 536/24.1, 435/320.1, 435/325, 435/419, 435/243, 435/69.1
• US Class Current: 435/69.1
• CPC Class Codes: C12N 15/815 for yeasts other than Saccharomyces
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione

Abstract

Polynucleotides that comprise regulatory regions of the yeast alcohol oxidase 1 promoter are provided. Isolated polynucleotides, recombinant polynucleotides, vectors, expression cassettes and transformed cells containing the regulatory regions are disclosed. Proteins produced by the transformed cells are also provided.

Claims

1. An isolated polynucleotide consisting of the nucleotide sequence as set forth in SEQ ID NO:

20.

5. A recombinant vector comprising a 5′

regulatory sequence operably linked to a heterologous coding region wherein the 5′

regulatory sequence consists of the isolated polynucleotide of any of claims 1-4.

6. An expression cassette comprising operably linked in 5′

to 3′

order a 5′

regulatory sequence, a heterologous coding region, and a termination sequence wherein said 5′

regulatory sequence consists of the isolated polynucleotide of any of claims 1-4.

7. A host cell comprising the vector of claim 5.

8. The host cell of claim 7 wherein said host cell is a yeast cell.

9. The host cell of claim 8 wherein said yeast cell is a methylotrophic yeast cell.

10. The host cell of claim 9 wherein said methylotrophic yeast cell is selected from the group of genera consisting or Hansenula, Cadida, Torulopsis, and Pichia.

11. The host cell of claim 10 wherein said methylotrophic yeast cell is a cell of Pichia pastoris.

12. A host cell comprising the expression cassette of claim 6.

13. The host cell of claim 12 wherein said host cell is a yeast cell.

14. The host cell of claim 13 wherein said yeast cell is a methylotrophic yeast cell.

15. The host cell of claim 14 wherein said methylotrophic yeast cell is selected from the group of genera consisting of Hansenula, Candida, Torulopsis and Pichia.

16. The host cell of claim 15 wherein said methylotrophic yeast cell is a cell of Pichia pastoris.

17. The host cell of claim 7 wherein said host cell expresses a protein encoded by said vector.

18. The host cell of claim 12 wherein said host cell expresses a protein encoded by said expression cassette.

19. A method for the production of a protein comprising growing the host cell of claim 17 under conditions where said host cell expresses the protein encoded by said vector and isolating the expressed protein.

20. A method for the production of a protein comprising growing the host cell of claim 18 under conditions where said host cell expresses the protein encoded by said vector and isolating the expressed protein.

2. An isolated polynucleotide consisting of the nucleotide sequence as set forth in SEQ ID NO:

27.

3. An isolated polynucleotide consisting of the nucleotide sequence as set forth in SEQ ID NO:

28.

4. An isolated polynucleotide consisting of the nucleotide sequence as set forth in SEQ ID NO:

29.