Sandhu P, Baba T, Guengerich F P
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Arch Biochem Biophys. 1993 Nov 1;306(2):443-50. doi: 10.1006/abbi.1993.1536.
The human cytochrome P450 (P450) 2C gene family is complex and heterologous expression methods are needed to facilitate the isolation of individual P450 proteins and the elucidation of their catalytic specificities. We prepared a series of constructs of P450 2C10 in the plasmid vector pCW, with modification of the 5' end of the coding sequence of the cDNA. Some were not expressed at all in Escherichia coli; two were expressed at levels of 5-20 nmol membrane-bound P450 (liter culture)-1--one (2C1028) with original codons 2-7 altered by substitution of the 5'-terminal sequence described by Barnes et al. (Barnes, H. J., Arlotto, M. P., and Waterman, M. R., Proc., Natl. Acad. Sci. USA 88, 5597-5601, 1991) and one (2C1029) with original codon 2 modified, codons 3-20 deleted, and alteration of the immediate downstream codons. In both cases the P450 2C10 proteins were found essentially only in the bacterial membranes. These proteins could be purified to a high degree by solubilization and a single DEAE chromatography step. Typical P450 Fe2+.CO absorption spectra were observed in the bacterial membranes and the purified preparations. The P450 2C1029 protein was found to have its N-terminal Met removed and the expected residues 2 (Ala)-24 were identified by amino acid sequence analysis. However, the other P450 (2C1028) was apparently blocked at the N-terminus. Three native P450 2C9/10 preparations isolated from human liver showed the expected sequences (beginning with Met) for at least the first 17 residues. The blocked N-terminus in the P450 2C1028 protein may be the result of the MALLLAVF sequence, which was also used in the expression of P450 3A4 and resulted in a blocked protein. Catalytic activities of P450 2C1028 and P450 2C1029 for tolbutamide hydroxylation were similar to those measured with purified liver P450 C29/10 in the presence of cytochrome b5, although the effect of cytochrome b5 did not always show the same pattern as with the isolated liver enzyme. The recombinant P450 2C10 enzymes did not catalyze (S)-mephenytoin 4'-hydroxylation.
人类细胞色素P450（P450）2C基因家族较为复杂，需要采用异源表达方法来促进单个P450蛋白的分离及其催化特异性的阐明。我们在质粒载体pCW中制备了一系列P450 2C10构建体，对cDNA编码序列的5'端进行了修饰。有些构建体在大肠杆菌中根本不表达；有两个构建体表达水平为5 - 20 nmol膜结合P450（每升培养物）-1，其中一个（2C1028）原始密码子2 - 7通过替换Barnes等人（Barnes, H. J., Arlotto, M. P., and Waterman, M. R., Proc., Natl. Acad. Sci. USA 88, 5597 - 5601, 1991）描述的5'端序列而改变，另一个（2C1029）原始密码子2被修饰，密码子3 - 20被删除，并改变了紧邻下游的密码子。在这两种情况下，P450 2C10蛋白基本上只存在于细菌膜中。这些蛋白可以通过溶解和单一的DEAE层析步骤高度纯化。在细菌膜和纯化制剂中观察到了典型的P450 Fe2 +.CO吸收光谱。发现P450 2C1029蛋白的N端甲硫氨酸被去除，通过氨基酸序列分析鉴定出预期的第2位（丙氨酸）至第24位残基。然而，另一种P450（2C1028）在N端显然被阻断。从人肝脏分离的三种天然P450 2C9/10制剂至少在前17个残基处显示出预期序列（以甲硫氨酸开始）。P450 2C1028蛋白中被阻断的N端可能是MALLLAVF序列的结果，该序列也用于P450 3A4的表达并导致蛋白被阻断。P450 2C1028和P450 2C1029对甲苯磺丁脲羟基化的催化活性与在细胞色素b5存在下用纯化的肝脏P450 C29/10测量的活性相似，尽管细胞色素b5的作用并不总是与分离的肝脏酶表现出相同的模式。重组P450 2C10酶不催化（S）-美芬妥因4'-羟基化。
