人白三烯A4水解酶cDNA在大肠杆菌中的表达。

Expression of human leukotriene A4 hydrolase cDNA in Escherichia coli.

作者信息

Minami M, Minami Y, Emori Y, Kawasaki H, Ohno S, Suzuki K, Ohishi N, Shimizu T, Seyama Y

机构信息

Department of Physiological Chemistry and Nutrition, Faculty of Medicine, University of Tokyo, Japan.

出版信息

FEBS Lett. 1988 Mar 14;229(2):279-82. doi: 10.1016/0014-5793(88)81140-2.

DOI:10.1016/0014-5793(88)81140-2

PMID:3278934

Abstract

The cDNA clone encoding human leukotriene A4 hydrolase was inserted into a vector pUC9 and expressed in Escherichia coli as a fusion protein containing the first 10 amino acid residues derived from a vector. The leukotriene A4 hydrolase activity was recovered in the soluble fraction of the transformants. The purified enzyme showed kinetic properties similar to the native enzyme, including inactivation by the substrate and sulfhydryl-modifying reagents. The results demonstrate that a protein with an Mr of 70,000 was expressed in Escherichia coli with a full enzyme activity and structural fidelity. Acquisition of the expression system makes it feasible to elucidate the reaction mechanism of the enzyme.

摘要

编码人白三烯A4水解酶的cDNA克隆被插入载体pUC9中，并在大肠杆菌中作为一种融合蛋白表达，该融合蛋白包含来自载体的前10个氨基酸残基。白三烯A4水解酶活性在转化体的可溶部分中得以恢复。纯化后的酶表现出与天然酶相似的动力学特性，包括被底物和巯基修饰试剂灭活。结果表明，一种分子量为70,000的蛋白质在大肠杆菌中得以表达，且具有完整的酶活性和结构保真度。该表达系统的获得使得阐明该酶的反应机制成为可能。