Methods for producing heterologous disulfide bond-containing polypeptides in bacterial cells
AssigneesBoard of Regents, The University of Texas System, Genentech, Inc.
InventorsGeorge Georgiou, Ji Oiu, Paul Bessette, James Swartz
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Abstract
Disclosed are methods and compositions for producing heterologous disulfide bond containing polypeptides in bacterial cells. In preferred embodiments the methods involve co-expression of a prokaryotic disulfide isomerase, such as DsbC or DsbG and a gene encoding a recombinant eukaryotic polypeptide. Exemplary polypeptides disclosed include tissue plasminogen activator.
Claims46
A process for producing a heterologous polypeptide in bacteria comprising:
- (a) culturing bacterial cells, wherein said bacterial cells comprise an isolated nucleic acid encoding a DsbG protein, nucleic acid encoding the heterologous polypeptide, a signal sequence for secretion of both the DsbG protein and the heterologous polypeptide, and separate inducible promoters for both the nucleic acid encoding the DsbG protein and the nucleic acid encoding the heterologous polypeptide, under conditions whereby expression of the nucleic acid encoding the DsbG protein is induced prior to induction of the expression of the nucleic acid encoding the heterologous polypeptide, and under conditions whereby either both the heterologous polypeptide and the DsbG protein are secreted into the periplasm of the bacteria or the heterologous polypeptide is secreted into the medium in which the bacterial cells are cultured;
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- (b) recovering the heterologous polypeptide from the periplasm or the culture medium.
A method of producing a biologically-active, soluble eukaryotic polypeptide having four or more disulfide bonds, comprising (a) expressing in a bacterial cell an isolated first DNA segment encoding a DsbG protein linked to a signal sequence and a second DNA segment encoding said eukaryotic polypeptide linked to a signal sequence under conditions effective to produce said eukaryotic polypeptide and (b) isolating said soluble eukaryotic polypeptide.
A method of producing a biologically-active, heterologous, eukaryotic polypeptide having four or more disulfide bonds, comprising (a) expressing in a bacterial cell an isolated first DNA segment encoding an E. coli DsbG protein operatively linked to a signal sequence, and a second DNA segment encoding said heterologous polypeptide operatively linked to a signal sequence, under conditions effective to produce said biologically-active, heterologous, eukaryotic polypeptide and (b) isolating said heterologous eukaryotic polypeptide.
A process for producing a correctly folded heterologous eukaryotic polypeptide having four or more disulfide bonds in a bacterial cell, said process comprising (a) expressing in said bacterial cell an isolated first DNA segment encoding an E. coli DsbG protein operatively linked to a signal sequence, and a second DNA segment encoding said heterologous polypeptide operatively linked to a signal sequence, under conditions effective to produce said correctly folded heterologous polypeptide and (b) isolating said heterologous eukaryotic polypeptide.
A method of producing a biologically-active, recombinant mammalian tissue plasminogen activator polypeptide, comprising (a) expressing in a bacterial cell an isolated first DNA segment encoding a DsbG protein operatively linked to a signal sequence and a second DNA segment encoding a recombinant mammalian tissue plasminogen activator operatively linked to a signal sequence under conditions effective to produce said recombinant mammalian tissue plasminogen activator polypeptide and (b) isolating said tissue plasminogen activator polypeptide.
A method of producing a biologically-active, recombinant mammalian pancreatic trypsin inhibitor polypeptide, comprising (a) expressing in a bacterial cell an isolated first DNA segment encoding an E. coli DsbG protein operatively linked to a signal sequence and a second DNA segment encoding a recombinant mammalian pancreatic trypsin inhibitor operatively linked to a signal sequence under conditions effective to produce said pancreatic trypsin inhibitor polypeptide and (b) isolating said pacreatic trypsin inhibitor polypeptide.
A bacterial expression system that expresses a DsbG protein, encoded by an isolated DNA, and a eukaryotic recombinant polypeptide having four or more disulfide bonds in a bacterial cell.
A bacterial expression system comprising a first expression unit comprising an isolated DNA segment encoding a disulfide isomerase operatively linked to a signal sequence and a second expression unit comprising a DNA segment encoding a recombinant polypeptide having at least about four disulfide bonds operatively linked to a signal sequence, wherein said expression system expresses a DsbG protein and said recombinant polypeptide.
A recombinant vector comprising a first transcriptional unit encoding an E. coli DsbG protein operatively linked to a signal sequence and said second transcriptional unit comprises a DNA segment encoding a mammalian tissue plasminogen activator or a mammalian pancreatic trypsin inhibitor.
Dependencies are inferred from the claim text.