Polypeptides and bacterial strains for increased protein production
View Patent ↗The invention provides polypeptides, nucleic acid sequences, bacterial strains, methods and kits for producing exogenous protein. More specifically, the invention provides RNase E polypeptides that posses impaired mRNA degrading activity and efficient rRNA and tRNA processing activity. These polypeptides may be utilized for producing exogenous protein.
1. A bacterial strain, the strain having a chromosomally integrated nucleotide sequence consisting essentially of a sequence that encodes an RNase E polypeptide with an amino acid sequence that is identical to SEQ ID NO:1, wherein the polypeptide possesses impaired ability to degrade mRNA.
2. The bacterial strain of claim 1 , wherein the polypeptide comprises post-translational modifications selected from the group consisting of glycosylation, phosphorylation, acetylation, alkylation, sulfation, ubiquitination, lipidation, carboxylation, biotinylation, and combinations thereof.
3. The bacterial strain of claim 1 , wherein the bacterial strain has impaired mRNA degrading activity compared to a bacterial strain having a nucleotide sequence that encodes a polypeptide with an amino acid sequence comprising SEQ ID NO:3.
4. The bacterial strain of claim 1 , wherein the bacterial strain is E. coli.
5. A method for enchancing production of an exogenous protein, the method comprising expressing the exogenous protein in a bacterial strain, the strain having a chromosomally integrated nucleotide sequence consisting essentially of a sequence that encodes an RNase E polypeptide with an amino acid sequence that is identical to SEQ ID NO:1, wherein the polypeptide possesses impaired ability to degrade mRNA.
6. The method of claim 5 , wherein the polypeptide comprises post-translational modifications selected from the group consisting of glycosylation, phosphorylation, acetylation, alkylation, sulfation, ubiquitination, lipidation, carboxylation, biotinylation, and combinations thereof.
7. The method of claim 5 , wherein the bacterial strain has impaired mRNA degrading activity compared to a bacterial strain having a nucleotide sequence that encodes a polypeptide with an amino acid sequence comprising SEQ ID NO:3.
8. The method of claim 5 , further comprising transforming the bacterial strain with an expression vector, the vector comprising a nucleotide sequence encoding at least one exogenous protein.
9. The method of claim 5 , further comprising transforming the bacterial strain with an exogenous inducible expression vector.
10. The method of claim 8 , wherein the bacterial strain is transformed by a method selected from the group consisting of electroporation, liposomal mediated transformation, calcium mediated transformation, and viral mediated transfection.
11. The method of claim 9 , wherein the exogenous inducible expression vector comprises a promoter selected from the group consisting of tac, trc, T7, lambda PL and araBAD.
12. The method of claim 5 , further comprising culturing the bacterial cells under conditions sufficient to permit expression of the exogenous protein.
13. The method of claim 12 , further comprising inducing the bacterial cells to express the exogenous protein.
14. The method of claim 5 , further comprising purifying the exogenous protein from the bacterial cells.
15. The method of claim 14 , wherein the exogenous protein is purified from the bacterial cells by a method selected from the group consisting of affinity purification, chromatography, solvent extraction, centrifugal sedimentation, and filtration.
16. A kit for producing an exogenous protein, the kit comprising:
(a) a bacterial strain, the strain having a chromosomally integrated nucleotide sequence consisting essentially of a sequence that encodes an RNase E polypeptide with an amino acid sequence that is identical to SEQ ID NO:1, wherein the polypeptide possesses impaired ability to degrade mRNA; and
(b) instructions for producing the exogenous protein.
17. The kit of claim 16 , wherein the cells comprising the bacterial strain are competent.
18. The kit of claim 16 , wherein the bacterial strain is E. coli.
19. The kit of claim 16 , further comprising sterile media for growing the bacterial cells.
20. The kit of claim 16 , further comprising an expression vector having an insertion site for the exogenous protein.
21. The kit of claim 20 , wherein the expression vector is inducible.
22. The kit of claim 21 , wherein the inducible expression vector comprises an inducible promoter.
23. The kit of claim 22 , wherein the inducible promoter is selected from the group consisting of tac, trc, T7, lambda PL and araBAD.