Method for producing therapeutic proteins in pichia pastoris lacking dipeptidyl aminopeptidase activity
AssigneeMerck Sharpe & Dohme Ltd.
InventorsStephen R. Hamilton, Terrance A. Stadheim
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Abstract
The present invention related to methods and compositions for producing therapeutic proteins in yeast cell lines, and in particular Pichia pastoris, lacking dipeptidyl aminopeptidase (DAP) activity. DAP activity has been eliminated by genetically modifying a Pichia pastoris cell line such that STE13 and DAP2 have been deleted.
Claims20
A method for producing a recombinant protein in a yeast host cell in which said host yeast cell lacks dipeptidyl aminopeptidase (DAP) activity comprising:
- a. transforming a genetically modified yeast cell in which DAP activity has been eliminated with a polynucleotide vector encoding the protein;
- b. culturing the transformed host cell in a medium under conditions which induce expression of the protein;
- and
- c. isolating the protein from the transformed host cell or culture medium.
A genetically modified yeast cell lacking dipeptidyl aminopeptidase (DAP) activity comprising a yeast cell line in which the genomic DNA encoding STE13 and DAP2 has been deleted or disrupted from the yeast cell genome.
A method for producing a therapeutic protein having an N-terminal recognition site Xaa-Pro or Xaa-Ala, where Xaa represents any one amino acid, in a yeast cell comprising:
- a. transforming a genetically modified yeast cell in which DAP activity has been eliminated with a polynucleotide vector encoding the therapeutic protein with the N-terminal recognition site selected from the group consisting of Xaa-Pro and Xaa-Ala;
- b. culturing the transformed host cell under conditions which induce expression of the therapeutic protein;
- and
- c. isolating the therapeutic protein from the transformed host cell.
Dependencies are inferred from the claim text.