Constructs and cells for enhanced protein expression
AssigneeMassachusetts Institute of Technology
InventorsKerry R. Love, J. Christopher Love, Charles Whittaker, Joseph Brady, Catherine Bartlett Matthews, Noelle Colant, Neil C. Dalvie
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Abstract
Described are expression constructs, cells, and methods of producing proteins in Pichia pastoris.
Claims159
An expression construct comprising an OLE1 promoter operably linked to a nucleic acid encoding a polypeptide comprising a signal sequence and a heterologous protein.
A methylotrophic cell expressing a heterologous protein, wherein the expression is under the control of an OLE1 promoter.
An expression construct comprising a DAS2 promoter operably linked to a nucleic acid encoding a polypeptide comprising a signal sequence and a heterologous protein and a targeting sequence for integration in a methylotrophic cell at a non-native locus.
A methylotrophic cell expressing a heterologous protein, wherein the expression is under the control of a DAS2 promoter integrated at a non-native locus.
An expression construct comprising an AOX1 promoter operably linked to a nucleic acid encoding a polypeptide comprising a signal sequence and a heterologous protein, the construct further comprising a targeting sequence for integration in a methylotrophic cell at a PIF1, OLE1, or DAS2 locus.
A methylotrophic cell expressing a heterologous protein, wherein the expression is under the control of an AOX1 promoter integrated at a PIF1, OLE1, or DAS2 locus.
An expression construct comprising a GAPDH promoter operably linked to a nucleic acid encoding a polypeptide comprising a signal sequence and a heterologous protein, the construct further comprising a targeting sequence for integration in a methylotrophic cell at an AOX1, PIF1, OLE1, or DAS2 locus.
A methylotrophic cell expressing a heterologous protein, wherein the expression is under the control of a GAPDH promoter integrated at an AOX1, PIF1, OLE1, or DAS2 locus.
An expression construct comprising a promoter operably linked to a nucleic acid encoding a polypeptide comprising a signal sequence and a heterologous protein, wherein the signal sequence is a signal sequence of KAR2, MSC1, TOS1, 2241, LHS1, TIF1, CTS1, or 5326.
A methylotrophic cell expressing a heterologous protein fused to a signal sequence, wherein the signal sequence is a signal sequence of KAR2, MSC1, TOS1, 2241, LHS1, TIF1, CTS1, or 5326.
A methylotrophic cell expressing a heterologous protein under the control of a promoter, wherein:
- (i) the promoter is an AOX1 promoter or a DAS2 promoter and/or the promoter is located at an AOX1 or DAS2 locus;
- (ii) mRNA encoding the heterologous protein comprises a Kozak sequence beginning at the −3 position relative to the translation start site;
- and/or
- (iii) a mRNA secondary structure of the mRNA encoding the heterologous protein has been reduced or eliminated relative to the endogenous mRNA encoding the heterologous protein.
An expression construct comprising a promoter operably linked to a nucleic acid encoding a polypeptide comprising a signal sequence and a heterologous protein, wherein:
- (i) the promoter is an AOX1 or DAS2 promoter and/or the construct further comprises a targeting sequence for integration in a methylotrophic cell at an AOX1 or DAS2 locus;
- (ii) the expression construct further comprises a Kozak sequence beginning at the −3 position relative to the translation start site of the nucleic acid encoding the polypeptide;
- and/or
- (iii) a mRNA secondary structure of the nucleic acid encoding a polypeptide has been reduced or eliminated relative to the endogenous nucleic acid encoding the polypeptide.
A method for preparing a transgene expression construct for expressing a heterologous protein in Pichia comprising:
- providing a nucleic acid encoding a heterologous protein;
- and
- (i) selecting a promoter that increases expression of genes of the Mut pathway upon integration;
- or
- (ii) selecting a targeting sequence for guided recombination into a locus, wherein insertion of the heterologous protein into the locus increases expression of genes of the Mut pathway;
- or
- (i) and (ii).
Dependencies are inferred from the claim text.