Overexpression and purification of the membrane-bound cytochrome P450 2B4.
Expression of the membrane-bound cytochrome P450 2B4 by the pLW01-P450 expression vector, which utilizes a T7 promoter, is markedly improved by employing Escherichia coli strain C41(DE3) [Miroux, B., and Walker, J. (1996) J. Mol. Biol 260, 289--298; Bridges, A., Gruenke, L., Chang, Y.-T., Vasker, I., Loew, G., and Waskell, L. (1998) J. Biol. Chem. 273, 17036--17049]. Using this expression system, it was possible to routinely obtain an average of 50--60 mg and as high as 100 mg of cyt P450 2B4 per liter of cell culture in volumes of 500 ml. An improved purification procedure for cyt P450 2B4 is also described which allows recovery of 30% of the expressed protein. It was possible in one step using B-PER reagent and polyoxyethylene-9-lauryl ether to both lyse the E. coli and solubilize the expressed cyt P450. Cyt P450 2B4 with a specific content of 17 nmol/mg protein and a single band on polyacrylamide gel electrophoresis was routinely isolated. The yield of cyt P450 from the improved purification procedure is twice that from the original procedure and the purity of the recovered protein typically has a specific content of 17 nmol cyt P450/mg of protein.
Department of Anesthesiology, University of Michigan, Ann Arbor, Michigan 48108, USA.
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Saribas
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Gruenke
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Waskell
D001189:Aryl Hydrocarbon Hydroxylases; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology* / Q000378:metabolism; D002847:Chromatography, Agarose; D002848:Chromatography, DEAE-Cellulose; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012685:Sepharose / Q000031:analogs & derivatives*; D013057:Spectrum Analysis; D013250:Steroid Hydroxylases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D011994:Recombinant Proteins; C027176:octyl-sepharose CL-4B; D012685:Sepharose; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C050356:steroid 15-alpha-hydroxylase