Saribas A S, Gruenke L, Waskell L
Department of Anesthesiology, University of Michigan, Ann Arbor, Michigan 48108, USA.
Protein Expr Purif. 2001 Mar;21(2):303-9. doi: 10.1006/prep.2000.1377.
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.
利用T7启动子的pLW01 - P450表达载体表达膜结合细胞色素P450 2B4时，通过使用大肠杆菌C41(DE3)菌株，其表达得到显著改善[米鲁克斯，B.，和沃克，J. (1996) 《分子生物学杂志》260, 289 - 298；布里奇斯，A.，格伦克，L.，张，Y.-T.，瓦斯克，I.，洛，G.，和瓦斯克尔，L. (1998) 《生物化学杂志》273, 17036 - 17049]。使用该表达系统，在500 ml体积的细胞培养物中，每升细胞培养物常规可平均获得50 - 60 mg、高达100 mg的细胞色素P450 2B4。还描述了一种改进的细胞色素P450 2B4纯化程序，该程序可回收30%的表达蛋白。使用B - PER试剂和聚氧乙烯 - 9 - 月桂基醚一步即可裂解大肠杆菌并溶解表达的细胞色素P450。常规分离得到比活性为17 nmol/mg蛋白且在聚丙烯酰胺凝胶电泳上呈单一条带的细胞色素P450 2B4。改进纯化程序的细胞色素P450产量是原程序的两倍，回收蛋白的纯度通常比活性为17 nmol细胞色素P450/mg蛋白。
