title: High-level expression of recombinant rabbit cytochrome P450 2E1 in Escherichia coli C41 and its purification.
abstract: Cytochrome P450 2E1 (CYP2E1) is of great interest because of its important role in the oxidation of numerous drugs and carcinogens. The yields of CYP2E1 obtained by the traditional recombinant expression systems have been relatively poor. We report here the development of a system for high-level expression of rabbit CYP2E1 in Escherichia coli strain C41 (DE3). Expression of the membrane-bound CYP2E1 by the pLW01-P450 expression plasmid, which utilizes a T7 promoter, is markedly improved by employing E. coli strain C41 (DE3). The pLW01/2E1 expression plasmid was successfully constructed and high-level expression of CYP2E1 was achieved, which ranged between 900 and 1400 nmol (liter culture)(-1). This yield was 9-14-fold higher than other reports of CYP2E1 expression in other E. coli strains. This system provides a highly efficient tool for expressing CYP2E1. An improved purification procedure for the expressed CYP2E1 involving chromatography on diethylaminoethyl cellulose (DE52), Reactive Red-agarose (type 1000-CL), and hydroxyapatite is also reported. This procedure allowed recovery of 45% of the expressed protein and CYP2E1 with a specific content of 14 nmol/mg protein, which showed a single band on a polyacrylamide gel stained with Coomassie brilliant blue.
authors.affiliation: Department of Pharmacology and Experimental Therapeutics, Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, 533 Bolivar Street, New Orleans, LA 70112, USA.
authors.forename: Dongmei
authors.initials: D
authors.lastname: Cheng
authors.forename: Rusty W
authors.initials: RW
authors.lastname: Kelley
authors.forename: George F
authors.initials: GF
authors.lastname: Cawley
authors.forename: Wayne L
authors.initials: WL
authors.lastname: Backes
mesh_terms: D000818:Animals; D002845:Chromatography / Q000379:methods; D019392:Cytochrome P-450 CYP2E1 / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013053:Spectrophotometry / Q000379:methods
chemical_list: D011994:Recombinant Proteins; D019392:Cytochrome P-450 CYP2E1