EXPRESSION OF MODIFIED HUMAN CYTOCHROME-P450 2E1 IN ESCHERICHIA-COLI, PURIFICATION, AND SPECTRAL AND CATALYTIC PROPERTIES
EXPRESSION OF MODIFIED HUMAN CYTOCHROME-P450 2E1 IN ESCHERICHIA-COLI, PURIFICATION, AND SPECTRAL AND CATALYTIC PROPERTIES
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DOI：
10.1006/abbi.1994.1280
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发表时间：
1994-07-01
影响因子：
3.9
通讯作者：
GUENGERICH, FP
中科院分区：
文献类型：
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作者：
GILLAM, EMJ;GUO, ZY;GUENGERICH, FP
人类细胞色素P450(P450)2E1因其在许多药物和致癌物的氧化中的作用而引起人们的兴趣。从人的肝脏中纯化蛋白质是困难的，我们报告了一种在大肠杆菌中相对高水平表达的系统的开发。采用聚合酶链式反应的方法，从肝组织中提取了一个cDNA，并构建了几个5‘末端修饰的变异体。对其中7个密码子的分析表明，当天然序列的前21个密码子被删除，并且所产生的N-末端Met之后的Trp被改变为Ala(GCT)时，发现表达水平最高。40nmoL膜结合的P450 2E1(升培养)(-1)的水平仅是常规恢复。重组蛋白P4502E1通过两步离子交换从菌膜中分离纯化，得率为80%。铁P450 2E1是在混合自旋状态下分离得到的。该酶在氯唑沙宗6-羟化反应中具有活性，人肝细胞色素b(5)的加入使底物的K-m降低，V-max增大。N-末端氨基酸序列分析得到了预期的前21个残基。该表达系统应有助于获得用于酶研究的人P450 2E1和抗体。(C)1994年学术出版社。
Human cytochrome P450 (P450) 2E1 is of interest because of its role in the oxidation of numerous drugs and carcinogens. The purification of the protein from human liver is difficult, and we report the development of a system for relatively high-level expression in Escherichia coli. A cDNA was prepared from liver cDNA by polymerase chain reaction methods and several variants with modified 5'-termini were constructed. Analysis of seven of these indicated that the highest levels of expression were found when the first 21 codons of the native sequence were deleted and the Trp immediately following the resulting N-terminal Met was changed to Ala (GCT). Levels of 40-nmol membrane-bound P450 2E1 (liter culture)(-1) mere routinely recovered. The recombinant P450 2E1 was purified to electrophoretic homogeneity from the bacterial membranes in two ion-exchange steps in >80% yield. Ferric P450 2E1 was isolated in a mixed spin state. The enzyme was active in chlorzoxazone 6-hydroxylation; the addition of human liver cytochrome b(5) lowered the K-m for the substrate and increased V-max. N-Terminal amino acid sequence analysis yielded the expected first 21 residues. The expression system should facilitate the availability of human P450 2E1 and antibodies for studies of the enzyme. (C) 1994 Academic Press, Inc.