Methods of producing two chain proteins in prokaryotic host cells
AssigneeGenentech, Inc.
InventorsKarthik VEERAVALLI, Rebekah MCKENNA
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Abstract
Provided herein are methods and host cells for producing a polypeptide containing two chains, such as an antibody, half-antibody, antibody fragment, or one-armed antibody. The methods and host cells allow for two-chain polypeptide production using expression of polynucleotides encoding the polypeptide chains from extra-chromosomal polynucleotide(s), and expression of one or more chaperone protein(s) (e.g., peptidyl-prolyl isomerases and/or protein disulfide oxidoreductases) from the host cell chromosome using non-native combination(s) of promoters and translational units encoding a chaperone protein.
Claims112
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 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;
- and
- (3) a third polynucleotide comprising a third translational unit encoding a chaperone protein selected from the group consisting of peptidyl-prolyl isomerases and protein disulfide oxidoreductases, wherein the third translational unit is part of the host cell chromosome, wherein the third translational unit is in operable combination with a 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 promoter is non-native to the host cell chromosome;
- and
- (b) recovering the biologically active polypeptide from the host cell.
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 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 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 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.
A prokaryotic host cell comprising a host cell chromosome, wherein the prokaryotic host cell comprises:
- (1) a first polynucleotide comprising a first translational unit encoding a peptidyl-prolyl isomerase, wherein the first translational unit is part of the host cell chromosome, wherein the first translational unit is in operable combination with a first promoter that is integrated in the host cell chromosome and drives transcription of the first translational unit, and wherein the combination of the first translational unit and the first promoter is non-native to the host cell chromosome;
- and
- (2) a second polynucleotide comprising a second translational unit encoding a protein disulfide oxidoreductase, wherein the second translational unit is part of the host cell chromosome, wherein the second translational unit is in operable combination with a second promoter that is integrated in the host cell chromosome and drives transcription of the second translational unit, and wherein the combination of the second translational unit and the second promoter is non-native to the host cell chromosome.
Dependencies are inferred from the claim text.