BACULOVIRUS EXPRESSION AND PURIFICATION OF HUMAN AND RAT CYTOCHROME-P450 2E1

High-level expression of human and rat cytochrome P450 2E1 (CYP2E1) wa s achieved using a baculovirus expression system. A full length cDNA e ncoding human CYP2E1 was cloned from a human liver cDNA library and se quenced using the dideoxy sequencing method. Insect cells were infecte d with the homologous recombinant baculoviruses containing the human a nd rat CYP2E1 cDNAs, respectively. The infected cells were harvested a t a time when 450-nm peak intensities were at a maximal level and ther e was no 420-nm peak observed in the reduced CO difference spectrum. B oth human and rat CYP2E1 were then purified to electrophoretic homogen eity by a relatively rapid and efficient procedure. The specific conte nts of the purified human and rat CYP2E1 were 13.8 and 17.0 nmol/mg pr otein, respectively. The lambda(max) of the reduced CO difference spec tra of both purified rat and human CYP2E1 was found to be 451.5 nm. Wh en the purified rat and human CYP2E1 were reconstituted with purified rat NADPH-P450 reductase and human cytochrome b(5), they were able to metabolize several known CYP2E1 substrates: chlorzoxazone, p-nitrophen ol, acetaminophen, and carbon tetrachloride. Interestingly, cytochrome b(5) markedly stimulated the CYP2E1-mediated two-electron oxidation o f the first three substrates, while it had almost no effect on the pre sumed one-electron reduction of carbon tetrachloride. (C) 1996 Academi c Press, Inc.