Purification and characterization of recombinant-expressed cytochrome P450 2C3 from Escherichia coli: 2C3 encodes the 6 beta-hydroxylase deficient form of P450 3b.
Purification and characterization of recombinant-expressed cytochrome P450 2C3 from Escherichia coli: 2C3 encodes the 6 beta-hydroxylase deficient form of P450 3b.
复制标题
从大肠杆菌中重组表达的细胞色素 P450 2C3 的纯化和表征：2C3 编码 P450 3b 的 6 β-羟化酶缺陷形式。
DOI：
10.1006/abbi.1993.1069
复制
发表时间：
1993
影响因子：
3.9
通讯作者：
Johnson,EF
中科院分区：
文献类型：
--
作者：
Richardson,TH;Hsu,MH;Kronbach,T;Barnes,HJ;Chan,G;Waterman,MR;Kemper,B;Johnson,EF
兔细胞色素 P450 2C3 在大肠杆菌中由其 cDNA 表达，这是一种嵌合酶，其中 2C3 的 N 端膜锚定序列的一部分被源自 P450 17α 的修饰序列取代。编码P450 17α N端的核苷酸序列先前被修饰以实现P450 17α在大肠杆菌中的高水平表达。大肠杆菌通过改变 P450 17α 的前八个密码子来反映高表达的第二个密码子偏好，并最大限度地减少相应 RNA 转录物形成稳定二级结构的可能性。修饰的 P450 2C3 的表达量>400 nmol/升培养物。分离出的 P450 2C3 具有明显的电泳均质性，且特定含量 >14 nmol P450/mg 蛋白质。当用 P450 还原酶和二月桂酰-L-α-卵磷脂重构时，纯化的大肠杆菌。大肠杆菌表达的 P450 2C3 催化黄体酮的 16α，但不催化 6β-羟基化。未修饰的 2C3 在 COS-1 细胞中的 cDNA 表达证实不存在可检测的 6β-羟化酶活性。此外，该酶在大肠杆菌中表达。大肠杆菌被变构效应子 5β-pregnane-3β,2Oα-diol 激活，产生的 Vmax10 min−1 和 Km20 μM，并且不受 16α-甲基孕酮抑制。这些结果表明，2C3 cDNA 编码 IIIvo/J 和 B/J 近交兔特有的酶形式，而不是大多数远交品系和一些近交品系中表达的第二种酶形式，其催化孕酮的高效 16α-和 6β-羟基化。我们的结果鉴定了 2C3 cDNA 编码的酶变体，并证明了大肠杆菌的实用性。大肠杆菌表达重组 P450 酶。
Rabbit cytochrome P450 2C3 was expressed from its cDNA inEscherichia colias 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 17α. The nucleotide sequence encoding the N-terminus of P450 17α was modified previously to achieve a high level of expression of P450 17α inE. coliby altering the first eight codons of P450 17α 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-α-lecithin, the purifiedE. coli-expressed P450 2C3 catalyzed 16α, but not 6β-hydroxylation of progesterone. Expression of unmodified 2C3 from its cDNA in COS-1 cells confirmed the absence of detectable 6β-hydroxylase activity. In addition, the enzyme expressed inE. coliis activated by the allosteric effector 5β-pregnane-3β,2Oα-diol, with a resultantVmax10 min−1andKm20 μM and is not inhibited by 16α-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 16α- and 6β-hydroxylation of progesterone. Our results have identified the enzyme variant encoded by the 2C3 cDNA and have demonstrated the utility ofE. colifor the expression of recombinant P450 enzymes.