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US 4,808,537 AGranted
Methanol inducible genes obtained from pichia and methods of use
AssigneePhillips Petroleum Company
InventorsDavid W. Stroman, Paul F. Brust, Steven B. Ellis, Thomas R. Gingeras, Michael M. Harpold, Juerg F. Tschopp
Life of this patent
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Abstract
A method for isolating and cloning methanol inducible genes from Pichia is taught. The regulatory regions are useful for the methanol regulated expression of heterologous genes.
Claims24
A method comprising:
- (a) culturing Escherichia coli NRRL B-15867 (LE392-pPG 6.0) in a nutrient medium;
- (b) disrupting the cultured cells;
- and
- (c) recovering plasmid pPG 6.0 from the disrupted cells.
1 more element
A method comprising:
- (a) culturing Escherichia coli NRRL B-15868 (LE392-pPG 4.0) in a nutrient medium;
- (b) disrupting the cultured cells;
- (c) recovering plasmid pPG 4.0 from the disrupted cells.
A method comprising:
- (a) culturing Escherichia coli NRRL B-15969 (LE392-pPG 4.8) in a nutrient medium;
- (b) disrupting the cultured cells;
- (c) recovering plasmid pPG 4.8 from the disrupted cells.
A method comprising:
- (a) culturing Escherichia coli NRRL B-15870 (LE392-pPC 15.0) in a nutrient medium;
- (b) disrupting the cultured cells;
- and
- (c) recovering plasmid pPC 15.0 from the disrupted cells.
1 more element
A method comprising:
- (a) culturing Escherichia coli NRRL B-15872 (LE392-pPC 6.7) in a nutrient medium;
- (b) disrupting the cultured cells;
- and
- (c) recovering plasmid pPC 6.7 from the disrupted cells.
1 more element
A method comprising:
- (a) culturing Escherichia coli NRRL B-15871 (LE392-pPC 8.3) in a nutrient medium;
- (b) disrupting the cultured cells;
- and
- (c) recovering plasmid pPC 8.3 from the disrupted cells.
1 more element
A method comprising:
- (a) culturing Escherichia coli NRRL B-15873 (MM294-pPC 8.0) in a nutrient medium;
- (b) disrupting the cultured cells;
- and
- (c) recovering plasmid pPC 8.0 from the disrupted cells.
1 more element
A method for isolating the p76 gene from a methanol assimilating yeast, said method comprising:
- (a) digesting total DNA with at least one restriction enzyme to give digested DNA;
- (b) size fractionating said digested DNA by agarose electrophoresis;
- (c) denaturating and binding the size fractionated DNA from step (b) to nitrocellulose filter to give bound DNA;
- (d) hybridizing said bound DNA with labeled p76 gene from Pichia pastoris;
- (e) identifying the unique size fragment of bound DNA which cross-hybridizes with the labeled p76 gene from P. pastoris;
- (f) size fractionating additional DNA which has been digested in accordance with step (a) to recover for cloning the unique size fragment of DNA identified in step (e).
3 more elements
A method for isolating the p72 gene from a methanol assimilating yeast, said method comprising:
- (a) digesting total DNA with at least one restriction enzyme to give digested DNA;
- (b) size fractionating said digested DNA by agarose electrophoresis;
- (c) denaturing and binding the size fractionated DNA from step (b) to nitrocellulose filter to give bound DNA;
- (d) hybridizing said bound DNA with labeled p72 gene from Pichia pastoris;
- (e) identifying the unique size fragment of bound DNA which cross-hybridizes with the labeled p72 gene from P. pastoris;
- (f) size fractionating additional DNA which has been digested in accordance with step (a) to recover for cloning the unique size fragment of DNA identified in step (e).
3 more elements
A method for isolating the p40 gene from a methanol assimilating yeast, said method comprising:
- (a) digesting total DNA with at least one restriction enzyme to give digested DNA;
- (b) size fractionating said digested DNA by agarose electrophoresis;
- (c) denaturing and binding the size fractionated DNA from step (b) to nitrocellulose filter to give bound DNA;
- (d) hybridizing said bound DNA with labeled p40 gene from Pichia pastoris;
- (e) identifying the unique size fragment of bound DNA which cross-hybridizes with the labeled p40 gene from P. pastoris;
- (f) size fractionating additional DNA which has been digested in accordance with step (a) to recover for cloning the unique size fragment of DNA identified in step (e).
3 more elements
Dependencies are inferred from the claim text.