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 containing an isolated nucleic acid sequence containing a P. pastoris alcohol dehydrogenase gene (ADH1) promoter having the nucleic acid sequence intervening nucleotides 1 to 1048 of SEQ ID NO: 18 operably linked to a structural gene which promoter directs the transcription of said structural gene under inducible conditions; wherein said inducible conditions comprise the expression of said structural gene operably linked to said promoter in the presence of ethanol or glycerol or a mixed feed comprising ethanol and glycerol.
2. The nucleic acid construct of claim 1 wherein the promoter is operably linked to a structural gene other than ADH1.
3. The nucleic acid construct of claim 1 which is contained on a plasmid.
4. The nucleic acid construct of claim 3 wherein said plasmid is an autonomously replicating plasmid or one which integrates into the chromosomal DNA of a yeast.
5. A yeast cell which contains a plasmid according to claim 3 .
6. The yeast cell of claim 5 which is a P. pastoris, Candida, Saccharomyces, Yarrowia, Kluyveromyces, Hansenula , or Schizosaccharomyces yeast cell.
7. The yeast cell of claim 6 which is polyploid.
8. The yeast cell of claim 7 which is a P. pastoris yeast.
9. A method of using a yeast cell according to claim 5 to produce a desired protein under the regulatory control of said ADH1 promoter, comprising growing the yeast cell under inducible conditions to produce the desired protein.
10. The method of claim 9 wherein said protein is multichain.
11. The method of claim 9 wherein said protein is a mammalian protein.
12. The method of claim 11 wherein said protein is an antibody or fragment thereof.
13. The method of claim 12 wherein said antibody or fragment is specific to a cytokine, or growth factor.
14. The method of claim 13 wherein the cytokine is selected from interleukins, an interferon, a tumor necrosis factor or a colony stimulating factor and the growth factor is a VEGF.
15. The method of claim 9 wherein said yeast cell is cultured in the presence of an amount of glycerol or ethanol or a mixed feed containing a sufficient amount of glycerol or ethanol to induce expression.
16. The method of claim 15 wherein said yeast cell contains at least one auxotrophic mutation, which requires supplementation for growth.
17. The method of claim 14 wherein the cytokine is selected from IL-2, IL-4, IL-6, IL-12, IL-13, interferon alpha, beta, or gamma, tumor necrosis factor or a colony stimulating factor.
18. The yeast cell of claim 5 which is a Pichia yeast cell.