title: Baculovirus-mediated expression of cytochrome P4502C8 and human NADPH-cytochrome P450 reductase: optimization of protein expression.
abstract: 1. High expression levels of cytochrome P450 (CYP) 2C8 and NADPH-cytochrome P450 oxidoreductase (OxR) in Spodoptera frugiperda (Sf21) cells have been achieved using the baculovirus expression system. 2. The baculovirus dual expression plasmid, pAcUW31, was used to insert CYP2C8 and OxR cDNAs downstream of the polyhedrin (polh) or p10 promoters, either separately or together, generating four recombinant baculoviruses; two expressing single proteins (CYP2C8 driven by the p10 promoter, bVp10.2C8 or OxR driven by the polh promoter, bVpolh.OxR) with another two coexpressing both CYP2C8 and OxR under reciprocal control of the polh and p10 promoters (bVpolh.OxR-p10.2C8 and bVpolh.2C8-p10.OxR). 3. High levels of singly expressed CYP2C8 and OxR were achieved from bVp10.2C8 and bVpolh.OxR, with levels of 0.7-1.2 nmol CYP/mg protein and 400-500 nmol cytochrome c reduced/min/mg protein respectively. 4. The two dual gene clones (bVpolh.OxR-p10.2C8 and bVpolh.2C8-p10.OxR) showed, in general, greater variation in CYP content and OxR activity than single gene clones. Screening was therefore necessary for the selection of dual gene clones expressing both proteins optimally. 5. Sf21 microsomes infected by selected dual gene clones were, on average, 14 times more active in tolbutamide hydroxylase activity than those expressing CYP2C8 alone, with a mean spectral CYP content of 79 pmol/mg cell lysate protein and a mean OxR level of 600 nmol/min/mg cell lysate protein.
authors.affiliation: Department of Clinical Pharmacology, Flinders Medical Centre, Adelaide, Australia.
authors.forename: C E
authors.initials: CE
authors.lastname: Ong
authors.forename: J O
authors.initials: JO
authors.lastname: Miners
authors.forename: D J
authors.initials: DJ
authors.lastname: Birkett
authors.forename: C R
authors.initials: CR
authors.lastname: Bhasker
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002478:Cells, Cultured; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006427:Hemin / Q000378:metabolism; D006801:Humans; D008861:Microsomes / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera / Q000235:genetics; D014162:Transfection / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D006427:Hemin; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C053553:tolbutamide 4-hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase