Kuo Lih-Ying, Hwang Guang-Yuh, Lai Yu-Jing, Yang Shin-Ling, Lin Long-Liu
Department of Biology, Tung-Hai University, 181 Taichung-Kan Road, Taichung, Taiwan.
Curr Microbiol. 2003 Jul;47(1):40-5. doi: 10.1007/s00284-002-3950-z.
For expression of Bacillus stearothermophilus NCIB 8924 leucine aminopeptidase II (LAP II) in Escherichia coli regulated by a T5 promoter, the gene was amplified by polymerase chain reaction and cloned into expression vector pQE-32 to generate pQE-LAPII. The His(6)-tagged enzyme was overexpressed in IPTG-induced E. coli M15 (pQE-LAPII) as a soluble protein and was purified to homogeneity by nickel-chelate chromatography to a specific activity of 425 U/mg protein with a final yield of 76%. The subunit molecular mass of the purified protein was estimated to be 44.5 kDa by SDS-PAGE. The temperature and pH optima for the purified protein were 60 degrees C and 8.0, respectively. Under optimal condition, the purified enzyme showed a marked preference for Leu- p-nitroanilide, followed by Arg- and Lys-derivatives. The His(6)-tagged enzyme was stimulated by Co(2+) ions, but was strongly inhibited by Cu(2+) and Hg(2+) and by the chelating agents, DTT and EDTA. The EDTA-treated enzyme could be reactivated with Co(2+) ions, indicating that it is a cobalt-dependent exopeptidase. Taking the biochemical characteristics together, we found that the recombinant LAP II exhibits no important differences from those properties described for the native enzyme.
为了在受T5启动子调控的大肠杆菌中表达嗜热栖热芽孢杆菌NCIB 8924亮氨酸氨肽酶II（LAP II），通过聚合酶链反应扩增该基因并克隆到表达载体pQE-32中，构建pQE-LAPII。带有His(6)标签的酶在IPTG诱导的大肠杆菌M15（pQE-LAPII）中作为可溶性蛋白过量表达，并通过镍螯合层析纯化至均一，比活性为425 U/mg蛋白，最终产率为76%。通过SDS-PAGE估计纯化蛋白的亚基分子量为44.5 kDa。纯化蛋白的最适温度和最适pH分别为60℃和8.0。在最佳条件下，纯化酶对亮氨酸对硝基苯胺表现出明显的偏好，其次是精氨酸和赖氨酸衍生物。带有His(6)标签的酶受到Co(2+)离子的刺激，但受到Cu(2+)、Hg(2+)以及螯合剂二硫苏糖醇（DTT）和乙二胺四乙酸（EDTA）的强烈抑制。经EDTA处理的酶可用Co(2+)离子重新激活，表明它是一种依赖钴的外肽酶。综合这些生化特性，我们发现重组LAP II与天然酶所描述的特性没有重要差异。
