Overexpression and purification of the membrane-bound cytochrome P4502B4

Expression of the membrane-bound cytochrome P450 2B4 by the pLW01-P450 expr ession vector, which utilizes a T7 promoter, is markedly improved by employ ing Escherichia coli strain C41(DE3) [Miroux, B., and Walker J, (1996) J. M el. 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 th is 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 vo lumes of 500 ml. An improved purification procedure for cyt P450 2B4 is als o described which allows recovery of 30% of the expressed protein. It was p ossible 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 2B 4 with a specific content of 17 nmol/mg protein and a single band on polyac rylamide gel electrophoresis was routinely isolated. The yield of cyt P450 from the improved purification procedure is twice that from the original pr ocedure and the purity of the recovered protein typically has a specific co ntent of 17 nmol cyt P450/mg of protein. (C) 2001 Academic Press