数字农科院2.0

Soluble expression and enzymatic activity evaluation of protease from reticuloendotheliosis virus

文献类型： 外文期刊

作者： Hu, Feng;Zhao, Yan;Qi, Xiaole;Cui, Hongyu;Gao, Yulong;Gao, Honglei;Liu, Changjun;Wang, Yongqiang;Zhang, Yanping;Li, Kai;Wang, Xiaomei;Wang, Yunfeng;Wang, Xiaomei;Wang, Yunfeng

作者机构：

关键词： Reticuloendotheliosis virus;Protease;Expression;Purification;Enzyme activity

期刊名称： PROTEIN EXPRESSION AND PURIFICATION

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收录情况： SCI

摘要： The protease (PR) encoded by most retroviruses is deeply involved in the lifecycle and infection process of retroviruses by possessing the specificity necessary to correctly cleave the viral polyproteins and host cell proteins. However, as an important representative of avian retroviruses, the enzymatic properties of PR from reticuloendotheliosis virus (REV) have not been clearly documented. The recombinant PR, its mutant fused with a His-tag, and its substrate p18-p30 fused with a GST-tag were expressed in the Escherichia coli system as soluble enzymes. The soluble PR and p18-p30 were purified using Ni-NTA His Bind Resin and Glutathione Sepharose 4B, respectively. The enzymatic activity of PR was analyzed using the substrate of p18-p30. The expressed prokaryotic protease has enzyme activity that is dependent on such conditions as temperature, pH, and ions, and its activity can be inhibited by caspase inhibitor and the divalent metal ions Ca2+ and Ni2+. In addition, the key role of the residue Thr (amino acids 28) for the enzymatic activity of PR was identified. Furthermore, the caspase inhibitor Z-VAD-FMK was confirmed to inhibit the PR enzymatic activity of REV. For the first time, the PR of REV was expressed in the soluble form, and the optimal enzymatic reaction system in vitro was developed and preliminarily used. This study provides essential tools and information for further understanding the infection mechanism of REV and for the development of antiviral drugs treating retroviruses. (C) 2015 Elsevier Inc. All rights reserved.

分类号： Q51

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