Modified bacterial protein expression system
View Patent ↗The present invention describes host cells for reliable, high yield recombinant protein production, including unstable proteins. The present host cell (e.g., a bacterial cell) is deficient in at least one protease (or a subunit of a protease) such as Clp or ClpP. The host cell may also contain an expression vector that encodes a protein or polypeptide for overexpression.
1. An engineered bacterium comprising at least one deficient protease, wherein the bacterium is engineered to disrupt and to express an exogenous peptide, wherein, prior to engineering, the bacterium is a protease deficient E. coli in a protease other than ClpP, wherein the engineered bacterium overexpresses the polypeptide at a level which is at least 2 fold greater compared to the level of the polypeptide expressed by said E. coli prior to disruption of ClpP, wherein said overexpression is induced at 37° C.
2. The engineered bacterium of claim 1 , wherein the E. coli is BL21(D3), wherein the overexpression level is greater than 3 fold, and wherein said overexpressed protein comprises a tag selected from the group consisting of SUMO, c-myc, biotin, polyhistidine and combinations thereof.
3. The engineered bacterium of claim 1 , wherein a gene encoding the protease is knocked out or knocked down in the engineered bacterium.
4. The engineered bacterium of claim 1 , wherein a gene encoding the protease is mutated or deleted in the engineered bacterium.
5. The engineered bacterium of claim 1 , further comprising deficient Lon, OmpT, FtsH, or combinations thereof.
6. The engineered bacterium of claim 2 , further comprising deficient Lon and deficient OmpT.
7. The engineered bacterium of claim 1 , wherein the bacterium comprises a selection marker.
8. The engineered bacterium of claim 7 , wherein the selection marker is selected from the group consisting of kanamycin, chloramphenicol, tetracyclin, ampicillin, vancomycin or erythromycin.
9. The engineered bacterium of claim 8 , wherein the selection marker is kanamycin.
10. The engineered bacterium of claim 2 , wherein the tag is polyhistidine.
11. The engineered bacterium of claim 1 , wherein the polypeptide is an antigen, an enzyme, a growth factor, a blood clotting factor, a hormone, or a transcription factor.
12. The engineered bacterium of claim 1 , wherein the polypeptide is a heterologous polypeptide.
13. The engineered bacterium of claim 1 , wherein the bacterium overexpresses a polypeptide at a level which is at least 5 fold of the level of the polypeptide produced by a bacterium not engineered with the deficient protease.
14. The engineered bacterium of claim 1 , wherein the bacterium overexpresses a polypeptide at a level which is at least 8 fold of the level of the polypeptide produced by a bacterium not engineered with the deficient protease.
15. A method for overexpressing a polypeptide, the method comprising the steps of: (a) culturing the engineered bacterium of claim 1 to produce the polypeptide; and (b) isolating the polypeptide.
16. A method for overexpressing a polypeptide in bacteria, the method comprising the steps of: (a) transforming the engineered bacterium of claim 1 with a nucleic acid sequence encoding the polypeptide; (b) culturing the bacterium to produce the polypeptide; and (c) isolating the polypeptide.
17. The method of any of claims 15 or 16 , wherein the polypeptide is a heterologous polypeptide.
18. A method for producing the engineered bacterium of claim 1 , wherein the protease is deleted by replacing the protease gene with a selection marker.
19. The method of claim 18 , wherein the selection marker is an antibiotic resistance gene.
20. The method of claim 19 , wherein the antibiotic is kanamycin, chloramphenicol, tetracyclin, ampicillin, vancomycin or erythromycin.
21. The method of claim 19 , wherein the antibiotic is kanamycin.