Yeast strains for protein production
AssigneeGlycoFi, Inc.
InventorJuergen Nett
Life of this patent
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Abstract
Method and system for expression systems, based on ade1 and ade2 auxotrophic strains of yeast and fungi, including P. pastoris are disclosed. The expression systems are useful for increased cellular productivity of transformed cell lines and for production of recombinant glycoproteins at industrial scale.
Claims33
An expression system comprising:
- (a) a host cell in which the endogenous gene encoding an auxotrophic selectable marker protein has been removed from the genome of the host cell;
- and
- (b) an integration vector comprising:
- (1) a nucleic acid having an open reading frame (ORF) encoding a function that is complementary to the function of the endogenous gene encoding the auxotrophic selectable marker protein and which is operably linked to a weak promoter, an attenuated endogenous or heterologous promoter, a cryptic promoter, a truncated endogenous or heterologous promoter, or no promoter;
- (2) a nucleic acid having an insertion site for the insertion of one or more expression cassettes comprising a nucleic acid encoding peptides, proteins, and/or functional nucleic acids of interest, and
- (3) a targeting nucleic acid that directs insertion of the integration vector into a particular location of the genome of the host cell by homologous recombination.
The expression system of claim 2 wherein the auxotrophic selectable marker protein is encoded by a gene selected from the group consisting of ADE, URA, and LYS.
A method for expressing a recombinant peptide, proteins, and/or nucleic acid of interest in a host cell comprising:
- (a) providing the host cell in which the endogenous gene encoding an auxotrophic selectable marker protein has been removed from the genome of the host cell;
- and
- (a) transforming the host cell with an integration vector comprising:
- (1) a nucleic acid having an open reading frame (ORF) encoding a function that is complementary to the function of the endogenous gene encoding the auxotrophic selectable marker protein and which is operably linked to a weak promoter, an attenuated endogenous or heterologous promoter, a cryptic promoter, a truncated endogenous or heterologous promoter, or no promoter;
- (2) a nucleic acid having one or more expression cassettes comprising a nucleic acid encoding peptides, proteins, and/or functional nucleic acids of interest, and
- (3) a targeting nucleic acid that directs insertion of the integration vector into a particular location of the genome of the host cell by homologous recombination,
- wherein the transformed host cell produces the recombinant peptide, protein and/or nucleic acid of interest.
The method of claim 14 wherein the auxotrophic marker is encoded by a gene selected from the group consisting of ADE, URA, and LYS.
An isolated nucleic acid comprising the ADE2 gene of Pichia pastoris.
A Pichia pastoris cell comprising a deletion or disruption of the endogenous ADE2 gene, or any other modification, rendering the ADE2 gene or Ade2p non-functional.
An isolated polypeptide comprising an amino acid sequence with 95% identity to the amino acid sequence shown in SEQ ID NO:61.
An expression system comprising:
- (a) a Pichia pastoris host cell in which the endogenous ADE2 gene encoding Ade2p has been removed from the genome of the host cell;
- and
- (b) an integration vector comprising:
- (1) a nucleic acid encoding the Ade2p;
- (2) a nucleic acid having an insertion site for the insertion of one or more expression cassettes comprising a nucleic acid encoding one or more heterologous peptides, proteins, and/or functional nucleic acids of interest, and
- (3) a targeting nucleic acid that directs insertion of the integration vector into a particular location of the genome of the host cell by homologous recombination.
A method for producing a recombinant Pichia pastoris host cell that expresses a heterologous peptide, proteins, and/or nucleic acid of interest comprising:
- (a) providing the host cell in which the endogenous ADE2 gene encoding an Ade2p has been removed from the genome of the host cell;
- and
- (a) transforming the host cell with an integration vector comprising:
- (1) a nucleic acid encoding the Ade2p;
- (2) a nucleic acid having one or more expression cassettes comprising a nucleic acid encoding one or more heterologous peptides, proteins, and/or functional nucleic acids of interest, and
- (3) a targeting nucleic acid that directs insertion of the integration vector into a particular location of the genome of the host cell by homologous recombination,
- wherein the transformed host cell produces the recombinant peptide, protein, and/or nucleic acid of interest.
Dependencies are inferred from the claim text.