The expression of a catalytically active cholesterol 7 alpha-hydroxylase cytochrome P450 in Escherichia coli.
The expression of a catalytically active cholesterol 7 alpha-hydroxylase cytochrome P450 in Escherichia coli.
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DOI：
10.1016/s0021-9258(18)54980-2
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发表时间：
1991-10
期刊：
影响因子：
--
通讯作者：
Y. Li;J. Chiang
中科院分区：
文献类型：
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作者：
Y. Li;J. Chiang
我们最近克隆了编码大鼠肝胆固醇7 α-羟化酶细胞色素P450（P450 c7）的全长cDNA（Li，Y. C.的方法，Wang，中国山杨D. P.，和Chiang，J. Y. L.（1990）J.Biol.Chem.265，12012-12019），其催化肝脏中胆汁酸合成的限速反应。利用聚合酶链反应技术，我们设计了两个P450 c7 cDNA。一个基因的第二个Met密码子缺失，第三个Thr密码子被Ala取代。另一个缺少氨基酸2-24的NH 2-末端疏水序列的密码子（P450 c7 delta 2-24）。将cDNA分别克隆到表达载体pKK 233 -2中，转化大肠杆菌。在用异丙基-β-D-硫代半乳糖苷诱导后，携带重组质粒的细菌表达了一种多肽，该多肽在免疫印迹中与抗胆固醇7 α-羟化酶的抗体反应。略微修饰的全长酶表达至总细菌裂解物的0.2%，并且位于膜部分中，而P450 c7 delta 2-24以高10倍的水平（2%）表达，其中85%在胞质溶胶中，其余与膜结合。我们已经纯化了P450 c7 delta 2-24，其显示了细胞色素P450的典型的还原CO差谱和在NADPH-细胞色素P450还原酶存在下的重构胆固醇7 α-羟化酶活性。与从大鼠肝微粒体分离的酶相比，P450 c7 delta 2-24对胆固醇具有相似的Km（24.6 μ M），但具有较低的Vmax（0.10 nmol/min）和较低的转换数（1.93 min-1）。纯化的P450 c7 delta 2-24具有独特的亲水性NH 2末端，并含有等量的单体和二聚体。这是第一份报告表明，基因工程细胞色素P450酶缺乏一个典型的NH 2-末端疏水序列主要是胞质和催化活性。
We have recently cloned a full-length cDNA encoding the rat hepatic cholesterol 7 alpha-hydroxylase cytochrome P450 (P450c7) (Li, Y. C., Wang, D. P., and Chiang, J. Y. L. (1990) J. Biol. Chem. 265, 12012-12019), which catalyzes the rate-limiting reaction of bile acid synthesis in the liver. By using the polymerase chain reaction, we have designed two P450c7 cDNAs. One has the second Met codon deleted and the third Thr codon replaced with an Ala. The other lacks codons for the NH2-terminal hydrophobic sequence of amino acids 2-24 (P450c7 delta 2-24). The cDNAs were separately cloned into the expression vector pKK233-2 and transformed into Escherichia coli. After induction with isopropyl-beta-D-thiogalactopyranoside, bacteria harboring recombinant plasmids expressed a polypeptide which reacted with the antibody against cholesterol 7 alpha-hydroxylase in immunoblots. The slightly modified full-length enzyme was expressed to 0.2% of the total bacterial lysate and was located in the membrane fraction, whereas P450c7 delta 2-24 was expressed at a 10-fold higher level (2%), of which 85% was in the cytosol and the remaining associated with the membranes. We have purified P450c7 delta 2-24 which showed a typical reduced-CO difference spectrum of cytochrome P450 and reconstituted cholesterol 7 alpha-hydroxylase activity in the presence of NADPH-cytochrome P450 reductase. P450c7 delta 2-24 has a similar Km for cholesterol (24.6 microM) but a lower Vmax (0.10 nmol/min) and a lower turnover number (1.93 min-1) as compared with the enzyme isolated from rat liver microsomes. The purified P450c7 delta 2-24 has an unique hydrophilic NH2 terminus and contains monomers and dimers in equal amounts. This is the first report demonstrating that a genetically engineered cytochrome P450 enzyme lacking a typical NH2-terminal hydrophobic sequence is mainly cytosolic and catalytically active.