Improvement in the expression of CYP2B6 by co-expression with molecular chaperones GroES/EL in Escherichia coli

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Abstract

Improvement of CYP2B6 expression was examined by co-expression with molecular chaperones GroES/EL. Although a CO-reduced difference spectrum was not detected in Escherichia coli transformed only by the CYP2B6-expressing vector, co-expression of GroES/EL resulted in high-level expression which reached over 2000 nmol P450/L. CYP2B6 was purified from the E. coli membrane with a high yield. Purified CYP2B6 showed 7-ethoxy-4-trifluoromethylcoumarin O-deethylase activity in a reconstitution system. This expression system would be useful for the production of large amounts of active CYP2B6 and for the detailed analysis of the enzyme.

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.

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