| abstract |
A process is provided for producing a heterologous polypeptide in bacteria. This process comprises, in a first step, culturing bacterial cells that lack their native pstS gene and comprise nucleic acid encoding a PstS variant having an amino acid variation within the phosphate-binding region of the corresponding native PstS, nucleic acid encoding a DsbA or DsbC protein, nucleic acid encoding the heterologous polypeptide, a signal sequence for secretion of both the DsbA or DsbC protein and the heterologous polypeptide, an inducible promoter for the nucleic acid encoding the DsbA or DsbC protein, and an alkaline phosphatase promoter for the nucleic acid encoding the heterologous polypeptide. The nucleic acid encoding a PstS variant is under the transcriptional control of the wild-type pstS gene promoter. The culturing step is conducted under conditions whereby expression of the nucleic acid encoding the DsbA or DsbC protein is induced prior to induction of the expression of the nucleic acid encoding the heterologous polypeptide, whereby the culturing takes place in a culture medium at a concentration of inorganic phosphate in the medium that during all phases of cell growth is above the level at which the cells are starved for phosphate, and whereby either both the heterologous polypeptide and the DsbA or DsbC 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. The second step of the process involves recovering the heterologous polypeptide from the periplasm or the culture medium. |