Regulatory region for heterologous gene expression in yeast
AssigneePhillips Petroleum Company
InventorsDavid W. Stroman, Paul F. Brust, Steven B. Ellis, Thomas R. Gingeras, Michael M. Harpold, Juerg F. Tschopp
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Abstract
Novel DNA sequences which are responsive to the presence of methanol, catabolite non-repressing carbon sources and carbon source starvation are provided. In addition, novel constructs including these DNA sequences, as well as transformed organisms therewith are provided. Processes for producing the DNA sequences and constructs of the invention are detailed. The production of polypeptide product under the control of the regulatory regions of the invention is demonstrated.
Claims98
An isolated DNA fragment derived from a yeast of the species Pichia pastoris comprising a regulatory region derived from a gene which codes for
- alcohol oxidase
- polypeptide p76 or
- polypeptide p40
- wherein said regulatory region is responsive to the presence of methanol in the culture medium with which a host organism for said DNA fragment is in contact and wherein said regulatory region is capable of controlling the transcription of messenger RNA when positioned at the 5' end of the DNA which codes for the production of said messenger RNA.
A DNA fragment in accordance with claim 19 further comprising an additional fragment positioned at the 3' end of the DNA which codes for the production of messenger RNA;
- wherein said additional fragment is selected from the group characterized by the restriction maps in FIG. 8 of the drawings.
An isolated DNA fragment comprising the 3' portion of the p40, p76 or alcohol oxidase genes derived from Pichia pastoris which fragment when positioned at the 3' end of a structural gene is capable of controlling the polyadenylation, termination of transcription and termination of translation of messenger corresponding to said structural gene and the 5' portion of the p40, p76 or alcohol oxidase genes derived from Pichia pastoris which fragment is responsive to the presence of methanol in the culture medium with which a host organism for said fragment is in contact and wherein said fragment is capable of controlling the transcription of messenger RNA when positioned at the 5' end of said structural gene.
An isolated gene derived from Pichia pastoris which upon expression codes for the production of biologically active alcohol oxidase or a biologically active sub-unit thereof.
An isolated Pichia pastoris gene coding for the production of polypeptide p76.
An isolated gene derived from Pichia pastoris which upon expression codes for the production of biologically active polypeptide p40 or a biologically active sub-unit thereof.
A methylotrophic yeast strain which has been transformed with a hybrid plasmid which plasmid comprises:
- (1) a5' regulatory region derived from a gene selected from the group consisting of the alcohol oxidase, p40 and p76 genes obtained from Pichia pastoris which regulatory region is responsive to methanol, operably linked to;
- (2) a structural gene which encodes a desired polypeptide and which transformed methylotrophic yeast is capable of expressing said desired polypeptide when cultured under suitable conditions for expression.
A process for preparing a desired polypeptide comprising:
- (1) transforming methylotrophic yeast strain with a hybrid plasmid which contains the 5' region of the p40, p76 or alcohol oxidase genes derived from Pichia pastoris, which regulatory region is operably linked to a gene encoding a desired polypeptide, and
- (2) culturing the transformed yeast strain in an amount of methanol sufficient to induce expression of the desired polypeptide.
The hybrid plasmid pPG 6.0.
The hybrid plasmid pPG 4.0.
The hybrid plasmid pPC 4.8.
The hybrid plasmid pPC 15.0.
The hybrid plasmid pPC 8.3.
The hybrid plasmid pPC 8.0.
The hybrid plasmid pPC 6.7.
The hybrid plasmid pSAOH 1.
The hybrid plasmid pSAOH 5.
The hybrid plasmid pSAOH 10.
The hybrid plasmid pTAFH 85.
The hybrid plasmid pT76H 1.
The hybrid plasmid pT76H 2.
The hybrid plasmid pT76H 3.
The hybrid plasmid pT76H 4.
The hybrid plasmid pTAO 13.
The hybrid plasmid pT76U 1.
An essentially pure culture of Escherichia coli NRRL B-15861;
- MCl061-pSAOH 1.
An essentially pure culture of Escherichia coli NRRL B-15862;
- MCl061-pSAOH 5.
An essentially pure culture of Escherichia coli NRRL B-15863;
- MCl061-pSAOH 10.
An essentially pure culture of Escherichia coli NRRL B-15864;
- MCl061-pTAFH 85.
An essentially pure culture of Escherichia coli NRRL B-15865;
- MCl061-pT76H 1.
An essentially pure culture of Escherichia coli NRRL B-15866;
- MCl060-pT76H 2.
An essentially pure culture of Escherichia coli NRRL B-18000;
- MCl061-pT76H 3.
An essentially pure culture of Escherichia coli NRRL B-18001;
- MCl061-pY76H 4.
An essentially pure culture of Escherichia coli NRRL B-18575;
- MCl061-pTAO 13.
An essentially pure culture of Escherichia coli NRRL B-18002;
- MCl061pT76U1.
An essentially pure culture of Escherichia coli NRRL B-15867;
- LE392-pPG 6.0.
An essentially pure culture of Escherichia coli NRRL B-15868;
- LE392-pPG 4.0.
An essentially pure culture of Escherichia coli NRRL B-15869;
- LE392-pPG 4.8.
An essentially pure culture of Escherichia coli NRRL B-15870;
- LE392-pPC 15.0.
An essentially pure culture of Escherichia coli NRRL B-15871;
- LE392-pPC 8.3.
An essentially pure culture of Escherichia coli NRRL B-15873;
- MM294-pPC 8.0.
An essentially pure culture of Escherichia coli NRRL B-15872;
- LE392-pPC 6.7.
Dependencies are inferred from the claim text.