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.
1. A nucleic acid construct comprising an isolated P. pastoris glycerol kinase (GUT1) gene promoter sequence comprising the nucleic acid sequence of SEQ ID NO:28 operably linked to a structural gene, wherein the promoter directs the transcription of the structural gene under inducible conditions.
2. The isolated nucleic acid construct of claim 1 , wherein said inducible conditions comprise the presence of glycerol or other mixed glycerol feed induction paradigm.
3. The nucleic acid construct of claim 1 , wherein the promoter is operably linked to a structural gene other than GUT1.
4. The nucleic acid construct of claim 1 , wherein the structural gene is a mammalian gene.
5. The nucleic acid construct of claim 1 , which is on a plasmid.
6. The nucleic acid construct of claim 1 , wherein said plasmid is an autonomously replicating plasmid or one which integrates into the chromosomal DNA of a yeast cell containing said plasmid.
7. A yeast cell which contains a plasmid according to claim 5 .
8. The yeast cell of claim 7 , which is a P. pastoris , Saccharomyces, Candida, Yarrowia, Kluyveromyces, Hansenula, or Schizzosaccharomyces yeast cell.
9. The yeast cell of claim 7 , which is polyploid.
10. The yeast cell of claim 9 , which is a P. pastoris yeast cell.
11. A stable culture containing a yeast cell according to claim 7 .
12. A method of producing a protein encoded by a structural gene under the regulatory control of the P. pastoris GUT1 promoter, comprising culturing the yeast cell according to claim 7 or the stable culture according to claim 11 under inducible conditions to produce the protein.
13. The method of claim 12 , wherein said protein is a multichain protein.
14. The method of claim 13 , wherein said protein is a mammalian protein.
15. The method of claim 14 , wherein said mammalian protein is an antibody is specific to a cytokine or growth factor.
16. The method of claim 15 , wherein said cytokine is an interferon such as interferon alpha, beta, or gamma, an interleukin such as IL-2, IL-4, IL-6, IL-12, or IL-13, a colony stimulating factor or a tumor necrosis factor and the growth factor is a VEGF.
17. The method of claim 12 , wherein said yeast cell is cultured in the presence of an amount of glycerol, or another carbon source or a mixed feed containing glycerol, sufficient to induce expression.
18. The method of claim 12 , wherein said yeast cell contains at least one auxotrophic mutation which requires supplementation for growth.
19. A fragment of the GUT1 promoter of claim 1 , which when operably linked with a promoter other than the GUT1 promoter renders the promoter glycerol inducible.