Efficient complementary DNA directed expression of human fetal liver cytochrome P450 (CYP3A7) in insect cells using baculovirus.
CYP3A7 is a form of cytochrome P450, which is expressed specifically in human fetal livers. NPVHF1, a recombinant baculovirus containing the entire coding region of CYP3A7, was constructed and infected to Spodoptera frugiperda (Sf9) cells. Upon infection with NPVHF1, the Sf9 cells expressed the CYP3A7 to the maximum content of 0.2 nmol per mg of whole cell lysates 72 hours after infection. A 5.5-fold expression level (1.1 nmol per mg of whole cell lysates) was attainable when cultured in the presence of externally added hemin. A catalytic activity of the CYP3A7 expressed in the Sf9 cells was confirmed by the umu gene expression mutation assay, in which aflatoxin B1 was activated to a mutagen by the expressed CYP3A7 in the presence of NADPH-cytochrome P450 reductase and cytochrome b5. From these results, it is concluded that the baculovirus expression system enables the high-level expression of CYP3A7 and will be a very useful tool for the characterization of CYP3A7.
Sakuma
T
T
Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Hokkaido University, Japan.
Kitamura
R
R
Yokoi
T
T
Kamataki
T
T
D016604:Aflatoxin B1 / Q000378:metabolism* / Q000633:toxicity*; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D016367:Baculoviridae; D002460:Cell Line; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism; D015786:Cytochromes b5 / Q000032:analysis / Q000096:biosynthesis; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005333:Fetus; D015870:Gene Expression / Q000187:drug effects; D006427:Hemin / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000196:embryology / Q000201:enzymology*; D008970:Molecular Weight; D009152:Mutagenicity Tests; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012486:Salmonella typhimurium / Q000187:drug effects; D018411:Spodoptera; D014162:Transfection
D018076:DNA, Complementary; D011994:Recombinant Proteins; D006427:Hemin; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D016604:Aflatoxin B1; D001189:Aryl Hydrocarbon Hydroxylases; C103123:CYP3A7 protein, human; D051544:Cytochrome P-450 CYP3A