US 11,667,944 B2
Methods and compositions for improved expression of recombinant proteins
Frederick R. Blattner, Madison, WI (US); Robert E. Novy, Verona, WI (US); David A. Frisch, Fitchburg, WI (US); Charles Landry, Fitchburg, WI (US); Hyunsic Choi, Madison, WI (US); Eric A. Steffen, Mount Horeb, WI (US); and John Brandon, Madison, WI (US)
Assigned to SCARAB GENOMICS, LLC, Madison, WI (US)
Appl. No. 16/644,444
Filed by SCARAB GENOMICS LLC, Madison, WI (US)
PCT Filed Sep. 4, 2018, PCT No. PCT/US2018/049422
§ 371(c)(1), (2) Date Mar. 4, 2020,
PCT Pub. No. WO2019/050872, PCT Pub. Date Mar. 14, 2019.
Claims priority of provisional application 62/554,443, filed on Sep. 5, 2017.
Prior Publication US 2020/0318150 A1, Oct. 8, 2020
Int. Cl. C12P 21/02 (2006.01); C12N 9/18 (2006.01); C12N 1/21 (2006.01); C12N 15/70 (2006.01); C12P 21/06 (2006.01); C07K 14/21 (2006.01); C07K 14/285 (2006.01); C07K 14/34 (2006.01); C12N 1/20 (2006.01)
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.