Improvement in the expression of CYP2B6 by co-expression with molecular chaperones GroES/EL in Escherichia coli
Section snippets
Chemicals
Bactotryptone, yeast extract, and bactopeptone were purchased from Becton Dickinson (Sparks, MD). Molecular chaperone plasmid pGro7 which expresses GroES/EL [13] was obtained from TAKARA BIO (Shiga, Japan). TOYOPEARL DEAE-650M and TOYOPEARL SP-550C were obtained from TOSOH (Tokyo, Japan). Hydroxyapatite was purchased from Bio-Rad (Hercules, CA). δ-Aminolevulinic acid was obtained from COSMO BIO (Tokyo, Japan). CYP2B6-expressed Supersomes were purchased from Gentest (Woburn, MA). NADPH-P450
Expression of CYP2B6 in E. coli
Fig. 1 shows the CO-reduced difference spectrum of E. coli cells transformed by the CYP2B6 expression vector without (A) or with (B) co-expressed molecular chaperones GroES/EL at 51 h after the addition of IPTG. In the absence of the molecular chaperone the characteristic peak of cytochrome P450 around 450 nm was not observed. In contrast, in the presence of the molecular chaperone, a high level of expression of the P450 molecule was spectrophotometrically detected. Fig. 2 shows the time-course
Conclusions
High-level expression of CYP2B6 in E. coli strain JM109 was achieved by co-expression with the molecular chaperones GroES/EL. The expression level of CYP2B6 exceeded 2000 nmol P450/L. CYP2B6 was purified in high yield from the membrane fraction. The purified CYP2B6 catalyzed 7-ethoxy-4-trifluoromethylcoumarin O-deethylation. It was considered that CYP2B6 expressed with molecular chaperones became tightly bound to E. coli membranes and that this would result in the high-level expression of the
Acknowledgments
The authors thank deeply Dr. Satoru Asahi for his helpful suggestions and discussions. The authors are grateful to Dr. Sumie Yoshitomi and Ms. Keiko Ikemoto for their technical guidance in detail throughout this study. The authors thank Dr. Norio Shimamoto, Dr. Kenji Okonogi, and Dr. Tetsuo Miwa for their encouragement. Authors express gratitude to Dr. F. W. Dahlquist for providing pCW expression vector.
References (21)
- et al.
Role of cDNA-expressed human cytochromes P450 in the metabolism of diazepam
Biochem. Pharmacol.
(1998) - et al.
Multiple forms of human P450 expressed in Saccharomyces cerevisiae. Systematic characterization and comparison with those of the rat
Biochem. Pharmacol.
(1996) - et al.
Expression of human cytochrome P450 2B6 in Escherichia coli: characterization of catalytic activity and expression levels in human liver
Arch. Biochem. Biophys.
(2000) - et al.
Expression of modified human cytochrome P450 1A2 in Escherichia coli: stabilization, purification, spectral characterization, and catalytic activities of the enzyme
Arch. Biochem. Biophys.
(1994) - et al.
A universal approach to the expression of human and rabbit cytochrome P450s of the 2C subfamily in Escherichia coli
Arch. Biochem. Biophys.
(1995) - et al.
Expression of modified human cytochrome P450 3A4 in Escherichia coli and purification and reconstitution of the enzyme
Arch. Biochem. Biophys.
(1993) - et al.
Development of bacterial expression system with high yield of CYP3A7, a human fetus-specific form of cytochrome P450
Biochem. Biophys. Res. Commun.
(2000) - et al.
Expression of modified human cytochrome P450 2E1 in Escherichia coli, purification, and spectral and catalytic properties
Arch. Biochem. Biophys.
(1994) - et al.
The carbon monoxide-binding pigment of liver microsomes. I. evidence for its hemoprotein nature
J. Biol. Chem.
(1964) - et al.
A truncation of 2B subfamily cytochromes P450 yields increased expression levels, increased solubility, and decreased aggregation while retaining function
Arch. Biochem. Biophys.
(2001)
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