在大肠杆菌中表达的重折叠人鼻病毒14型2A蛋白酶的酶学特性分析
Enzymatic characterization of refolded human rhinovirus type 14 2A protease expressed in Escherichia coli.
作者信息
Wang Q M, Johnson R B, Cox G A, Villarreal E C, Churgay L M, Hale J E
机构信息
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, USA.
出版信息
J Virol. 1998 Feb;72(2):1683-7. doi: 10.1128/JVI.72.2.1683-1687.1998.
Reported here is the production of recombinant human rhinovirus 14 (HRV14) 2A protease from bacterial cells transformed with a heat-inducible plasmid containing the HRV14 2A cDNA sequence. Overexpressed 2A protein partitioned into the inclusion bodies was solubilized in urea and then refolded in the presence of Zn2+ Transition metals were required for the restoration of 2A protease activity as a structural component, but appeared to be inhibitory if added exogenously once the enzyme was refolded. Based on the cleavage specificity studies, a colorimetric assay was developed for the highly purified HRV14 2A protease. A peptide with the sequence RKGDIKSY-p-nitroanilide was found to be cleaved by the 2A protease with a k(cat)/Km ratio of approximately 335 M(-1)s(-1), which allows its activity to be measured continuously with a spectrophotometer or a microplate reader.
本文报道了用含有HRV14 2A cDNA序列的热诱导质粒转化的细菌细胞生产重组人鼻病毒14（HRV14）2A蛋白酶的过程。过量表达的2A蛋白在包涵体中被溶解于尿素中，然后在Zn2+存在下进行重折叠。过渡金属作为结构成分是恢复2A蛋白酶活性所必需的，但如果在酶重折叠后外源添加则似乎具有抑制作用。基于裂解特异性研究，开发了一种用于高度纯化的HRV14 2A蛋白酶的比色测定法。发现序列为RKGDIKSY-p-硝基苯胺的肽被2A蛋白酶裂解，其k(cat)/Km比值约为335 M(-1)s(-1)，这使得可以用分光光度计或微孔板读数器连续测量其活性。
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