title: cDNA-directed expression of human cytochrome P450 CYP1A1 using baculovirus. Purification, dependency on NADPH-P450 oxidoreductase, and reconstitution of catalytic properties without purification.
abstract: A recombinant baculovirus containing the human cytochrome P450 (CYP) 1A1 cDNA was constructed and used to express CYP1A1 in Spodoptera frugiperda (SF9) insect cells (0.14 +/- 0.04 nmol/mg protein, 53 +/- 14 nmol/liter, N = 30). The enzyme represented approximately 1% of total cellular protein and was partially purified by a three-column procedure to a specific content of 5.0 nmol/mg protein. Catalytic activity was reconstituted with both the purified enzyme using lipid and NADPH-P450 oxidoreductase, and the SF9 insect cell membrane fraction without purification using NADPH-P450 oxidoreductase and small amounts of detergent. Catalytic activity of the enzyme after reconstitution was optimum using molar ratios of CYP1A1 to NADPH-P450 oxidoreductase of 1:8. Cytochrome b5 had no additional stimulating effect. The enzyme metabolized substrates characteristic for CYP1A1:benzo[a]pyrene (4.0 +/- 0.3 nmol/min/nmol CYP), 7-ethoxy-4-trifluoromethyl- coumarin (36 +/- 2), ethoxyresorufin (37 +/- 1), but not pentoxyresorufin (0.77 +/- 0.02). Recombinant baculovirus expresses the highest amounts of all expression systems published to date of catalytically active CYP1A1. Because human CYP1A1 has never been isolated in a catalytically active state from human tissue, nor has recombinant unmodified human CYP1A1, this system is an excellent alternative for the isolation and characterization of this CYP.
authors.affiliation: National Institutes of Health, National Cancer Institute, Bethesda, MD 20892, USA.
authors.forename: J T
authors.initials: JT
authors.lastname: Buters
authors.forename: M
authors.initials: M
authors.lastname: Shou
authors.forename: J P
authors.initials: JP
authors.lastname: Hardwick
authors.forename: K R
authors.initials: KR
authors.lastname: Korzekwa
authors.forename: F J
authors.initials: FJ
authors.lastname: Gonzalez
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera / Q000201:enzymology / Q000821:virology*; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D007527:Isoenzymes; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase