Patent
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.
1. An E. coli strain, wherein:
i) the strain lacks the activity of a thioredoxin reductase encoded by trxB due to genetic mutation of trxB;
ii) the strain lacks the activity of a thioredoxin 1 encoded by trxA due to genetic mutation of trxA;
iii) the strain lacks the activity of a glutathione reductase encoded by gor due to genetic mutation of gor; and
iv) the strain expresses a mutated AhpC protein, wherein the mutated AhpC protein has glutathione reductase activity.
2. The E. coli strain of claim 1 , wherein the E. coli strain further comprises a gene encoding a protein of interest.
3. The E. coli strain of claim 2 , wherein the protein of interest is selected from the group consisting of: an antibody, a fragment thereof, and an antibody light chain from an IgG.
4. The E. coli strain of claim 1 , wherein the mutated AphC protein lacks peroxyreductase activity, and wherein the mutated AphC protein comprises a sequence of SEQ ID NO: 5.
5. The E. coli strain of claim 1 , wherein the E. coli strain has reduced protein expression or mRNA expression of the wild type TrxA, TrxB, and Gor relative to a control E. coli strain.
6. The E. coli strain of claim 1 , wherein the E. coli strain expresses a mutated TrxA protein, and wherein the mutated TrxA protein lacks the activity of the corresponding wild-type protein.
7. The E. coli strain of claim 1 , wherein the E. coli strain expresses a mutated TrxB protein, and wherein the mutated TrxB protein lacks the activity of the corresponding wild-type TrxB protein.
8. The E. coli strain of claim 1 , wherein the E. coli strain expresses a mutated Gor protein, and wherein the mutated Gor protein lacks the activity of the corresponding wild type Gor protein.
9. The E. coli strain of claim 2 , wherein the gene encoding the protein of interest is operably linked to an inducible promoter.
10. The E. coli strain of claim 9 , wherein the inducible promoter is a T7 promoter.
11. The E. coli strain of claim 1 , wherein the mutated AphC protein is encoded by a mutated ahpC gene, and wherein the expression of the ahpC gene is controlled by a Pc0 promoter.
12. The E. coli strain of claim 1 , wherein the E. coli strain is a K-12 strain.
13. 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 due to genetic mutation of trxB;
ii) lacks thioredoxin 1 activity due to genetic mutation of trxA;
iii) lacks the activity of a glutathione reductase encoded by gor due to genetic mutation of gor; and
iv) expresses a mutated AhpC protein, wherein the mutated AhpC protein has glutathione reductase activity.
14. The method of claim 13 , wherein the mutated AphC protein lacks peroxyreductase activity, and wherein the mutated AphC protein comprises a sequence of SEQ ID NO: 5.
15. The method of claim 13 , wherein the E. coli strain further expresses a wild-type AhpC protein comprising a sequence of SEQ ID NO: 3.
16. The method of claim 13 , wherein the E. coli strain has reduced protein expression or mRNA expression of the wild type TrxA, TrxB, and Gor relative to a control E. coli strain.
17. The method of claim 13 , wherein the E. coli strain expresses a mutated TrxA protein, and wherein the mutated TrxA protein lacks the activity of the corresponding wild-type protein.
18. The method of claim 13 , wherein the E. coli strain expresses a mutated TrxB protein, and wherein the mutated TrxB protein lacks the activity of the wild-type TrxB protein.
19. The method of claim 13 , wherein the E. coli strain expresses a mutated Gor protein, and wherein the mutated Gor protein lacks the activity of the wild type Gor protein.
20. The method of claim 13 , wherein the E. coli strain contains a null mutation in one or both of trxB and trxA.
21. The method of claim 13 , wherein the protein of interest is selected from the group consisting of: an IgG, a light chain from an IgG, and a heavy chain from an IgG.
22. A kit comprising the E. coli of claim 1 , wherein the kit further comprises a growth medium.
23. The kit of claim 22 , wherein the kit further comprises a plasmid encoding a protein of interest.