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Abstract

cDNA-directed expression of human cytochrome P450 CYP1A1 using baculovirus. Purification, dependency on NADPH-P450 oxidoreductase, and reconstitution of catalytic properties without purification.

J T Buters, M Shou, J P Hardwick, K R Korzekwa and F J Gonzalez
Drug Metabolism and Disposition July 1995, 23 (7) 696-701;
J T Buters
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M Shou
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J P Hardwick
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K R Korzekwa
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F J Gonzalez
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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.

 

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Drug Metabolism and Disposition
Vol. 23, Issue 7
1 Jul 1995
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Abstract

cDNA-directed expression of human cytochrome P450 CYP1A1 using baculovirus. Purification, dependency on NADPH-P450 oxidoreductase, and reconstitution of catalytic properties without purification.

J T Buters, M Shou, J P Hardwick, K R Korzekwa and F J Gonzalez
Drug Metabolism and Disposition July 1, 1995, 23 (7) 696-701;

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Abstract

cDNA-directed expression of human cytochrome P450 CYP1A1 using baculovirus. Purification, dependency on NADPH-P450 oxidoreductase, and reconstitution of catalytic properties without purification.

J T Buters, M Shou, J P Hardwick, K R Korzekwa and F J Gonzalez
Drug Metabolism and Disposition July 1, 1995, 23 (7) 696-701;
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