Elsevier

Biochemical Pharmacology

Volume 55, Issue 8, 15 April 1998, Pages 1315-1325
Biochemical Pharmacology

Research papers
High Catalytic Activity of Human Cytochrome P450 Co-expressed with Human NADPH-Cytochrome P450 Reductase in Escherichia coli

https://doi.org/10.1016/S0006-2952(97)00643-6Get rights and content

Abstract

Forms of human cytochrome P450 (P450 or CYP), such as CYP1A1, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4, were expressed or co-expressed together with human NADPH-P450 reductase in Escherichia coli. When P450 was expressed alone in E. coli, the expression level of holo-P450 ranged from 310 to 1620 nmol/L of culture. The expression level of holo-P450 decreased by co-expression with the reductase, and the level ranged from 66 to 381 nmol/L of culture. The expression level of the reductase varied depending on the forms of P450 co-expressed, and ranged from 204 to 937 U/L of culture. We assayed the catalytic activity of P450 using E. coli cells disrupted by freeze–thaw. When co-expressed with the reductase, human P450 catalyzed the oxidation of representative substrates at efficient rates. The rates appeared comparable to the reported activities of P450 in a reconstituted system containing purified preparations of P450 and the reductase.

Section snippets

Materials

NADP+, glucose-6-phosphate, and glucose-6-phosphate dehydrogenase were obtained from Oriental Yeast; 7-ethoxyresorufin, 7-hydroxyresorufin, 7-ethoxycoumarin, 7-hydroxycoumarin, coumarin, and propranolol from the Aldrich Chemical Co.; 11β- and 6β-hydroxytestosterone from Steraroid, Inc.; tolbutamide and 4-hydroxytolbutamide, 4-nitrophenol, 1,2-dihydroxy-4-nitrobenzene, o-hydroxybenzamide, aniline hydrochloride, p-aminophenol, taxol, taxotere, testosterone, and phenobarbital sodium from Wako Pure

Expression of P450 and the Reductase

We constructed expression plasmids with an insert of P450 cDNA alone for nine forms of P450 or together with the reductase cDNA. The plasmids thus constructed were introduced into E. coli DH5α. The expression level of holo-P450 determined by carbon monoxide difference spectra is shown in Table 1. Introduction of the plasmid into E. coli resulted in the expression of large amounts of the hemoprotein, while a wide variation in the expression level was observed. The expression level ranged from

Discussion

In the present study, we were able to express human P450 together with the reductase in E. coli, and found that P450s expressed in E. coli showed catalytic activities. Compared with the Km and Vmax values of human P450s reported thus far, P450s expressed in E. coli did not necessarily show values identical to those obtained using human liver microsomes (Table 3). The reasons for the discrepancy may be explained as follows. First, liver microsomes contain many forms of P450, and the Km and Vmax

Acknowledgements

This study was supported, in part, by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan, and, in part, by the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Drug ADR Relief, R & D Promotion and Product Review of Japan.

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