US 12,291,712 B2
Methods of producing two chain proteins in prokaryotic host cells
Karthik Veeravalli, San Francisco, CA (US); and Rebekah McKenna, Hayward, CA (US)
Assigned to Genentech, Inc., South San Francisco, CA (US)
Filed by Genentech, Inc., South San Francisco, CA (US)
Filed on May 4, 2021, as Appl. No. 17/307,877.
Application 17/307,877 is a continuation of application No. PCT/US2019/059661, filed on Nov. 4, 2019.
Claims priority of provisional application 62/755,915, filed on Nov. 5, 2018.
Prior Publication US 2021/0261973 A1, Aug. 26, 2021
Int. Cl. C12N 15/70 (2006.01); C07K 16/00 (2006.01); C07K 16/22 (2006.01); C07K 16/24 (2006.01); C12N 9/02 (2006.01); C12N 9/90 (2006.01)
CPC C12N 15/70 (2013.01) [C07K 16/00 (2013.01); C07K 16/22 (2013.01); C07K 16/244 (2013.01); C12N 9/0004 (2013.01); C12N 9/90 (2013.01); C12Y 502/01008 (2013.01); C07K 2317/14 (2013.01); C07K 2317/31 (2013.01)] 15 Claims
 
1. A method of producing a polypeptide comprising two chains in a prokaryotic host cell comprising a host cell chromosome, the method comprising:
(a) culturing the host cell to express the two chains of the polypeptide in a culture medium under conditions suitable for expression of the two chains of the polypeptide, whereby upon expression the two chains fold and assemble to form a biologically active polypeptide in the host cell;
wherein the host cell is an Escherichia coli (E. coli) strain, and wherein the host cell comprises:
(1) a first polynucleotide comprising a first translational unit encoding a first chain of the polypeptide;
(2) a second polynucleotide comprising a second translational unit encoding a second chain of the polypeptide, wherein the first and second polynucleotides are part of one or more extra-chromosomal polynucleotides;
(3) a third polynucleotide comprising a third translational unit encoding a protein disulfide oxidoreductase, wherein the third translational unit is part of and native to the host cell chromosome, wherein the third translational unit is in operable combination with a first promoter that is integrated in the host cell chromosome and drives transcription of the third translational unit, and wherein the combination of the third translational unit and the first promoter is non-native to the host cell chromosome; and
(4) a fourth polynucleotide comprising a fourth translational unit encoding a peptidyl-prolyl isomerase, wherein the fourth translational unit is part of and native to the host cell chromosome, wherein the fourth translational unit is in operable combination with a second promoter that is integrated in the host cell chromosome and drives transcription of the fourth translational unit, and wherein the combination of the fourth translational unit and the second promoter is non-native to the host cell chromosome; and
(b) recovering the biologically active polypeptide from the host cell,
wherein:
the protein disulfide oxidoreductase is E. coli DsbC, wherein the first promoter is a Pho promoter that drives transcription of the third translational unit when phosphate in the culture medium has been depleted, wherein the peptidyl-prolyl isomerase is E. coli FkpA, and wherein the second promoter is a CP25 promoter; or
the protein disulfide oxidoreductase is E. coli DsbC, wherein the first promoter is a Pho promoter that drives transcription of the third translational unit when phosphate in the culture medium has been depleted, wherein the peptidyl-prolyl isomerase is E. coli FkpA, and wherein the second promoter is a Pho promoter that drives transcription of the fourth translational unit when phosphate in the culture medium has been depleted.