Cytochrome P450 2A1, 2E1, and 2C9 cDNA-expression by insect cells and partial purification using hydrophobic chromatography.
High-level expression of three cloned cytochrome P450 enzymes was accomplished using the baculovirus-insect cell expression system. The amount of enzyme expression was enhanced by cell infections in the presence of medium-supplements containing hemin and by growth in suspension cultures. Human cytochromes P450 2E1 and 2C9 and rat cytochrome P450 2A1 were partially purified from cell extracts using hydrophobic interaction and hydroxyapatite chromatography. The resulting enzymes were of estimated molecular masses similar to those reported previously and analyzed by PAGE. Reconstitution of enzyme activity resulted when the enzymes were incubated together with NADPH-cytochrome P450 reductase, phospholipid, NADPH, and appropriate substrates. The cytochrome P450 activity of the partially purified enzymes was comparable to that of the corresponding enzymes expressed in the vaccinia virus-Hep G2 system. These results provide evidence for a general means of obtaining cytochrome P450 enzymes for mechanistic, immunochemical, and biophysical investigations.
Grogan
J
J
Laboratory of Molecular Carcinogenesis, National Cancer Institute, Bethesda, MD 20892, USA.
Shou
M
M
Andrusiak
E A
EA
Tamura
S
S
Buters
J T
JT
Gonzalez
F J
FJ
Korzekwa
K R
KR
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D016367:Baculoviridae / Q000235:genetics; D002384:Catalysis; D002460:Cell Line; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002845:Chromatography; D019392:Cytochrome P-450 CYP2E1; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D018076:DNA, Complementary / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D005822:Genetic Vectors; D006427:Hemin / Q000494:pharmacology; D006801:Humans; D010089:Oxidoreductases, N-Demethylating / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012967:Sodium Dodecyl Sulfate; D018411:Spodoptera; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D014407:Tumor Cells, Cultured; D014616:Vaccinia virus / Q000235:genetics
D018076:DNA, Complementary; D011994:Recombinant Proteins; D012967:Sodium Dodecyl Sulfate; D006427:Hemin; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D019392:Cytochrome P-450 CYP2E1; D001189:Aryl Hydrocarbon Hydroxylases; D039181:Steroid 16-alpha-Hydroxylase; D010089:Oxidoreductases, N-Demethylating