Construction of a human cytochrome P450 1A1:Rat NADPH-cytochrome P450 reductase fusion protein cDNA and expression in Escherichia coli, purification, and catalytic properties of the enzyme in bacterial cells and after purification
Construction of a human cytochrome P450 1A1:Rat NADPH-cytochrome P450 reductase fusion protein cDNA and expression in Escherichia coli, purification, and catalytic properties of the enzyme in bacterial cells and after purification
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DOI：
10.1006/abbi.1996.0224
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发表时间：
1996-06-01
影响因子：
3.9
通讯作者：
Guengerich, FP
中科院分区：
文献类型：
--
作者：
Chun, YJ;Shimada, T;Guengerich, FP
对质粒（pCW）进行修饰以编码由人细胞色素P450（P450）1A 1的完整序列组成的融合蛋白（仅第二个氨基酸改变）在通过Ser-Thr接头连接到大鼠NADPH-P450还原酶的从氨基酸57开始的部分的N-末端部分，该质粒用于在大肠杆菌DH 5 α细胞中表达融合蛋白，并使用DEAE和2 ′，5 ′-ADP琼脂糖层析从去污剂溶解的细菌膜中纯化蛋白。纯化的融合蛋白催化苯并[a]芘3-羟基化、7-乙氧基试卤灵O-脱乙基化和唑胺6-羟基化，在添加NADPH-P450还原酶、细胞色素B（5）或磷脂的情况下，催化活性没有增加。融合蛋白还可以将电子转移到细胞色素c和B（5），但不能转移到P450 1A 2，用纯化的融合蛋白和在细菌细胞中起作用的融合蛋白形成相同的苯并[a]芘氧化产物，人P450 1A 1融合蛋白对几种底物的催化活性明显低于用大鼠P450 1A 1构建的类似融合蛋白，P450 1A 1，与所报道的大鼠和人P450 1A 1酶催化活性的差异一致，纯化的融合蛋白在没有添加NADPH-P450还原酶的情况下也氧化（+）-和（-）-苯并[a]芘7，8-二氢二醇和8种芳基和杂环胺为对生殖毒性的产物，人融合蛋白在细菌细胞内的催化活性的证明表明了利用这种细胞系统生产人P450代谢物的前景。(C)出版社：Academic Press，Inc.
A plasmid (pCW) was modified to code for a fusion protein consisting of the complete sequence of human cytochrome P450 (P450) 1A1 (with only the second amino acid changed) in the N-terminal portion connected by a Ser-Thr linker to the portion of rat NADPH-P450 reductase beginning at amino acid 57, This plasmid was used to express the fusion protein in Escherichia coli DH5 alpha cells and the protein was purified from detergent-solubilized bacterial membranes using DEAE and 2',5'-ADP agarose chromatography. The purified fusion protein catalyzed benzo[a]pyrene 3-hydroxylation, 7-ethoxyresorufin O-deethylation, and zoxazolamine 6-hydroxylation, Catalytic activity was not increased in the presence of added NADPH-P450 reductase, cytochrome b(5), or phospholipid, The fusion protein could also transfer electrons to cytochromes c and b(5) but not P450 1A2, The same oxidation products of benzo[a]pyrene were formed with the purified fusion protein and the fusion protein functioning in bacterial cells, The catalytic activity of the human P450 1A1 fusion protein toward several substrates is markedly less than that of a similar fusion protein constructed with rat P450 1A1, in line with the reported differences in catalytic activities of the rat and human P450 1A1 enzymes, The purified fusion protein also oxidized (+)- and (-)-benzo[a]pyrene 7,8-dihydrodiols and eight aryl and heterocyclic amines to ge-notoxic products, in the absence of added NADPH-P450 reductase, The demonstration of catalytic activities of the human fusion protein within bacterial cells suggests the prospect of utilizing such cellular systems for production of human P450 metabolites. (C) 1996 Academic Press, Inc.