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