Bacterial hosts for recombinant protein expression
AssigneePelican Technology Holdings, Inc.
InventorsAnthony Farina, Cory M. Schwartz, Torben Bruck, Russell Coleman, Diane M. Retallack
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Abstract
The present invention provides recombinant gram-negative host cells that do not degrade protease-sensitive recombinant proteins yet grow to high cell density, methods for the use of these host cells to produce high-quality recombinant proteins, including antibodies and antibody fragments, at high yield, as well as compositions and methods relating to periplasmic expression of recombinant proteins or polypeptides of interest in host cells.
Claims44
A recombinant gram-negative Pseudomonas fluorescens host cell for recombinant protein expression, wherein the host cell is:
- (a) deficient in a first protease activity, wherein the first protease activity is tail-specific protease activity, wherein the deficient first protease activity results from a mutation in a gene encoding a Prc1 tail-specific protease having the amino acid sequence set forth as SEQ ID NO:
- 33, a Prc1 tail-specific protease related protein having an amino acid sequence at least 90% similar to SEQ ID NO:
- 33, a Prc2 tail-specific protease having the amino acid sequence set forth as SEQ ID NO:
- 35, or a Prc2 tail-specific protease related protein having an amino acid sequence at least 90% similar to SEQ ID NO:
- 35;
- and,
- (b) deficient in a second protease activity, wherein the second protease activity is murein DD-endopeptidase activity, wherein the deficient second protease activity results from a mutation in at least one gene encoding a murein DD-endopeptidase;
- and wherein the host cell further:
- (c) is deficient in at least one additional protease activity, wherein the deficient additional protease activity results from a mutation in at least one gene encoding an additional protease, wherein the additional protease is different from the proteases of (a) and (b);
- (d) is deficient in one or more autolytic factor activity, wherein the deficient autolytic factor activity results from a mutation in at least one gene encoding an autolytic factor;
- (e) overexpresses one or more inactivated protease;
- (f) overexpresses one or more folding modulator;
- or
- (g) any combination of (c), (d), (e) and (f).
A recombinant gram-negative Pseudomonas fluorescens host cell, wherein the host cell is:
- (a) deficient in a first protease activity, wherein the deficient first protease activity results from mutations in
- i. a gene encoding a Prc1 tail-specific protease having the amino acid sequence set forth as SEQ ID NO:
- 33;
- and,
- ii. a gene encoding a Prc2 tail-specific protease having the amino acid sequence set forth as SEQ ID NO:
- 35;
- and
- (b) deficient in a second protease activity, wherein the deficient second protease activity results from a mutation in a gene encoding a MepM1 murein DD-endopeptidase having the amino acid sequence set forth as SEQ ID NO:
- 1;
- and additionally wherein the host cell is further:
- (c) deficient in one additional protease activity, wherein the deficient additional protease activity results from a mutation of a gene encoding a serralysin precursor having the amino acid sequence set forth as SEQ ID NO:
- 9;
- (d) deficient in one or more autolytic factor activity, wherein the deficient autolytic factor activity results from a mutation in at least one gene encoding an autolytic factor independently selected from:
- a linear gramicidin synthase subunit D, or a hemolysin precursor;
- (e) overexpresses a mutant periplasmic serine endoprotease;
- (f) overexpresses a disulfide isomerase folding modulator-having at least 60% similarity or at least 60% identity to an amino acid sequence set forth as SEQ ID NO:
- 27;
- or
- (g) any combination of (c), (d), (e) and (f).
Dependencies are inferred from the claim text.