Baculovirus expression and purification of human and rat cytochrome P450 2E1.
High-level expression of human and rat cytochrome P450 2E1 (CYP2E1) was achieved using a baculovirus expression system. A full length cDNA encoding human CYP2E1 was cloned from a human liver cDNA library and sequenced using the dideoxy sequencing method. Insect cells were infected with the homologous recombinant baculoviruses containing the human and rat CYP2E1 cDNAs, respectively. The infected cells were harvested at a time when 450-nm peak intensities were at a maximal level and there was no 420-nm peak observed in the reduced CO difference spectrum. Both human and rat CYP2E1 were then purified to electrophoretic homogeneity by a relatively rapid and efficient procedure. The specific contents of the purified human and rat CYP2E1 were 13.8 and 17.0 nmol/mg protein, respectively. The lambda(max) of the reduced CO difference spectra of both purified rat and human CYP2E1 was found to be 451.5 nm. When the purified rat and human CYP2E1 were reconstituted with purified rat NADPH-P450 reductase and human cytochrome b5, they were able to metabolize several known CYP2E1 substrates: chlorzoxazone, p-nitrophenol, acetaminophen, and carbon tetrachloride. Interestingly, cytochrome b5 markedly stimulated the CYP2E1-mediated two-electron oxidation of the first three substrates, while it had almost no effect on the presumed one-electron reduction of carbon tetrachloride.
Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle 98195-7610, USA.
W
W
Chen
R M
RM
Peter
S
S
McArdle
K E
KE
Thummel
R O
RO
Sigle
S D
SD
Nelson
D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D019392:Cytochrome P-450 CYP2E1 / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D018076:DNA, Complementary; D006427:Hemin / Q000032:analysis; D006801:Humans; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry; D018411:Spodoptera; D013379:Substrate Specificity; D014162:Transfection
D018076:DNA, Complementary; D011994:Recombinant Proteins; D006427:Hemin; D019392:Cytochrome P-450 CYP2E1