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US 11,407,975 B2Granted
E. coli strains having an oxidative cytoplasm
AssigneeSutro Biopharma, Inc.
InventorDaniel Groff
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Abstract
This disclosure provides an E. coli strain, which lacks thioredoxin reductase activity encoded by trxB and thioredoxin 1 activity encoded by trxA, and glutathione reductase activity encoded by gor. Said E. coli strain expresses a mutated AhpC protein having glutathione reductase activity and a cytosolic prokaryotic disulfide isomerase. The E. coli strain has an oxidative cytosol and can be used to efficiently produce proteins having disulfide bonds.
Claims29
An E. coli strain, wherein:
- i) the strain lacks the activity of a thioredoxin reductase encoded by trxB;
- ii) the strain lacks the activity of a thioredoxin 1 encoded by trxA;
- iii) the strain lacks the activity of a glutathione reductase encoded by gor;
- iv) the strain expresses a mutated AhpC protein, wherein the mutated AhpC protein has glutathione reductase activity but lacks peroxyreductase activity;
- and
- v) the strain expresses a cytosolic prokaryotic disulfide isomerase.
3 more elements
A method for expressing soluble, recombinant proteins of interest in E. coli bacterial strains comprising the steps of:
- culturing an E. coli bacterial strain comprising an oxidizing cytosol and an expression cassette for expressing a protein of interest under conditions that permit expression of the protein of interest as a soluble protein, wherein the strain:
- i) lacks thioredoxin reductase activity;
- ii) lacks thioredoxin 1 activity;
- iii) lacks the activity of a glutathione reductase encoded by gor;
- iv) expresses an ahpC gene that has been mutated such that an enzyme expressed from the ahpC gene has glutathione reductase activity but lacks peroxyreductase activity;
- and
- v) expresses a gene encoding a cytosolic prokaryotic disulfide isomerase.
4 more elements
Dependencies are inferred from the claim text.