cDNA-directed expression of human cytochrome P450 CYP3A4 using baculovirus.
A recombinant baculovirus containing the human CYP3A4 cDNA was constructed and used to express CYP3A4 in SF9 insect cells (0.46 +/- 0.13 nmol/mg protein, 103 +/- 29 nmol/liter, N = 15). The enzyme represented approximately 2-3% of total cellular protein and could be purified by a two-column procedure to a specific content of 12.7 nmol/mg protein. Catalytic activity of the purified enzyme after reconstitution was optimum using molar ratios of CYP3A4 to cytochrome b5 to NADPH-P450 oxidoreductase of 1:3:20, respectively. The enzyme metabolized cortisol, erythromycin, testosterone, and (R)-warfarin. Recombinant baculovirus expresses the highest amounts of all expression systems published to date of catalytically intact CYP3A4. This system is an excellent alternative for the isolation and characterization of P450 forms from human liver.
Buters
J T
JT
National Institutes of Health, National Cancer Institute, Bethesda, MD 20892.
Korzekwa
K R
KR
Kunze
K L
KL
Omata
Y
Y
Hardwick
J P
JP
Gonzalez
F J
FJ
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007313:Insecta; D006899:Mixed Function Oxygenases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D011995:Recombination, Genetic; D013379:Substrate Specificity
D018076:DNA, Complementary; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human