Consortium of fold-catalyzing proteins increases soluble expression of cyclohexanone monooxygenase in recombinant Escherichia coli
- 257 Downloads
The cyclohexanone monooxygenase (CHMO) gene of Acinetobacter sp. NCIMB 9871 was simultaneously expressed with the genes encoding molecular chaperones and foldases in Escherichia coli. While the expression of the CHMO gene alone resulted in the formation of inclusion bodies, coexpression of the chaperone or foldase genes remarkably increased the production of soluble CHMO enzyme in recombinant E. coli. Furthermore, it was found that molecular chaperones were more beneficial than foldases for enhancing active CHMO enzyme production. The recombinant E. coli strain simultaneously expressing the genes for CHMO, GroEL/GroES and DnaK/DnaJ/GrpE showed a specific CHMO activity of 111 units g−1 cell protein, corresponding to a 38-fold enhancement in CHMO activity compared with the control E. coli strain expressing the CHMO gene alone.
KeywordsMolecular Chaperone Coli Strain Soluble Expression Folding Pathway Villiger Oxidation
We are grateful to Professor Jon D. Stewart (University of Florida, Gainesville, Fla.) for his kind donation of the plasmid pMM4. This work was supported by the Center for Advanced Bioseparation Technology and the Ministry of Education, through the BK21 program.
- Amrein KE, Takacs B, Steiger M, Molnos J, Flint NA, Burn P (1995) Purification and characterization of recombinant human p50csk protein-tyrosine kinase from and Escherichia coli expression system overproducing the bacterial chaperones GroES and GroEL. Proc Natl Acad Sci USA 92:1048–1052PubMedGoogle Scholar
- Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 27:680–685Google Scholar
- Nishihara K, Kanemori M, Kitagawa M, Yanagi H, Yura T (1998) Chaperone coexpression plasmids: differential and synergistic roles of DnaK-DnaJ-GrpE and GroEL-GroES in assisting folding of an allergen of Japanese cedar pollen Cryj2, in Escherichia coli. Appl Environ Microbiol 64:1694–1699PubMedGoogle Scholar
- Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.Google Scholar
- Shin CS, Hong MS, Shin HC, Lee JW (2001) High-level production of recombinant human IFN-α2a with co-expression of tRNAArg(AGG/AGA) in high-cell-density cultures of Escherichia coli. Biotechnol Bioprocess Eng 6:301–305Google Scholar
- Stewart JD (1998) Cyclohexanone monooxygenase: a useful reagent for asymmetric Bayer–Villiger reactions. Curr Org Chem 2:195–216Google Scholar
- Thomas JG, Baneyx F (1996) Protein misfolding and inclusion body formation in recombinant Escherichia coli cells overexpressing heat-shock proteins. J Biol Chem 271:1141–1147Google Scholar