CPC C12P 21/06 (2013.01) [C07K 14/21 (2013.01); C07K 14/285 (2013.01); C07K 14/34 (2013.01); C12N 1/20 (2013.01); C12N 9/18 (2013.01); C07K 2319/02 (2013.01)] | 17 Claims |
1. A method for extracting a recombinant polypeptide from the periplasm of an Escherichia coli host cell, the host cell comprising:
a first nucleotide sequence encoding the recombinant polypeptide fused to a signal sequence that directs transfer of the recombinant polypeptide to the periplasm of the E. coli host cell, said first nucleotide sequence operably linked to an expression control sequence comprising an inducible promoter; and
a second nucleotide sequence encoding an E. coli phospholipase, said second nucleotide sequence operably linked to a heterologous expression control sequence comprising an inducible promoter, wherein said first and second nucleotide sequences are different;
the method comprising:
(a) culturing the E. coli host cell in a medium under conditions permitting co-expression of the recombinant polypeptide and the phospholipase in the E. coli host cell;
(b) contacting the E. coli host cell comprising the expressed recombinant polypeptide and the expressed phospholipase with an extraction buffer comprising a chelating agent for at least 6 hours and less than 24 hours at a temperature between 20° C. and 30° C. to release the recombinant polypeptide from the periplasm of the E. coli host cell into the extraction buffer; and
(c) collecting the recombinant polypeptide from the extraction buffer.
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