Richardson T H, Hsu M H, Kronbach T, Barnes H J, Chan G, Waterman M R, Kemper B, Johnson E F
Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, California 92037.
Arch Biochem Biophys. 1993 Jan;300(1):510-6. doi: 10.1006/abbi.1993.1069.
Rabbit cytochrome P450 2C3 was expressed from its cDNA in Escherichia coli as a chimeric enzyme in which a portion of the N-terminal membrane anchor sequence of 2C3 was replaced with a modified sequence derived from P450 17 alpha. The nucleotide sequence encoding the N-terminus of P450 17 alpha was modified previously to achieve a high level of expression of P450 17 alpha in E. coli by altering the first eight codons of P450 17 alpha to reflect second codon preferences for high expression and to minimize the potential for the formation of a stable secondary structure of the corresponding RNA transcript. The modified P450 2C3 was expressed at > 400 nmol/liter of culture. P450 2C3 was isolated to apparent electrophoretic homogeneity and a specific content > 14 nmol P450/mg protein. When reconstituted with P450 reductase and dilauroyl-L-alpha-lecithin, the purified E. coli-expressed P450 2C3 catalyzed 16 alpha, but not 6 beta-hydroxylation of progesterone. Expression of unmodified 2C3 from its cDNA in COS-1 cells confirmed the absence of detectable 6 beta-hydroxylase activity. In addition, the enzyme expressed in E. coli is activated by the allosteric effector 5 beta-pregnane-3 beta,20 alpha-diol, with a resultant Vmax = 10 min-1 and Km = 20 microM and is not inhibited by 16 alpha-methylprogesterone. These results indicate that the 2C3 cDNA encodes an enzymatic form characteristic of IIIvo/J and B/J inbred rabbits rather than a second enzymatic form expressed in most outbred and some inbred strains that catalyzes both high efficiency 16 alpha- and 6 beta-hydroxylation of progesterone. Our results have identified the enzyme variant encoded by the 2C3 cDNA and have demonstrated the utility of E. coli for the expression of recombinant P450 enzymes.
兔细胞色素P450 2C3在大肠杆菌中由其cDNA表达为一种嵌合酶，其中2C3的N端膜锚定序列的一部分被源自细胞色素P450 17α的修饰序列所取代。编码细胞色素P450 17α N端的核苷酸序列先前已被修饰，通过改变细胞色素P450 17α的前八个密码子以反映高表达的第二个密码子偏好，并使相应RNA转录本形成稳定二级结构的可能性最小化，从而在大肠杆菌中实现细胞色素P450 17α的高水平表达。修饰后的细胞色素P450 2C3在每升培养物中表达量>400 nmol。细胞色素P450 2C3被分离至表观电泳纯，比含量>14 nmol细胞色素P450/mg蛋白质。当与细胞色素P450还原酶和二月桂酰-L-α-卵磷脂重构时，纯化的大肠杆菌表达的细胞色素P450 2C3催化孕酮的16α-羟基化，但不催化6β-羟基化。细胞色素P450 2C3未修饰的cDNA在COS-1细胞中的表达证实不存在可检测到的6β-羟化酶活性。此外，在大肠杆菌中表达的该酶被变构效应物5β-孕烷-3β,20α-二醇激活，结果Vmax = 10 min-1，Km = 20 μM，且不被16α-甲基孕酮抑制。这些结果表明，2C3 cDNA编码的是IIIvo/J和B/J近交系兔特有的酶形式，而不是大多数远交系和一些近交系中表达的另一种酶形式，后者可高效催化孕酮的16α-和6β-羟基化。我们的结果鉴定了由2C3 cDNA编码的酶变体，并证明了大肠杆菌在重组细胞色素P450酶表达中的实用性。
