Specific and efficient cleavage of fusion proteins by recombinant plum pox virus NIa protease.
Specific and efficient cleavage of fusion proteins by recombinant plum pox virus NIa protease.
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重组李痘病毒NIa蛋白酶特异性高效切割融合蛋白
DOI：
10.1016/j.pep.2007.10.008
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发表时间：
2008-02
影响因子：
1.6
通讯作者：
Xie Q
中科院分区：
文献类型：
--
作者：
Zheng N;Pérez Jde J;Zhang Z;Domínguez E;Garcia JA;Xie Q
位点特异性蛋白酶是用于从融合靶蛋白去除融合标签的最常用的酶。马铃薯Y病毒科（Potyviridae）的核包涵体蛋白a（Nuclear Inclusion Protein a，NIa）蛋白酶由于其高活性和序列识别的严格性而成为研究的热点。来自烟草蚀纹病毒（TEV）和番茄脉斑驳病毒（TVMV）的NIa蛋白酶已被证明在体外成功地加工重组蛋白。利用重组PPV（plumepox virus）NIa蛋白酶体外加工具有人工切割位点的融合蛋白。研究了催化能力和影响因素（盐、温度、蛋白酶抑制剂、洗涤剂和变性剂）等特性。从大肠杆菌中表达和纯化的重组PPV NIa蛋白酶证明了重组GFP和SARS-CoV核衣壳蛋白的有效和特异性加工，其中PPV NIa蛋白酶的位点F（NV V H Q A）人工插入融合标签和靶蛋白之间。其催化能力与TVMV和TEV NIa蛋白酶相似。重组PPV NIa蛋白酶在约30 °C下达到其最大蛋白水解活性。盐浓度和仅一种测试的蛋白酶抑制剂对PPV NIa蛋白酶的蛋白水解活性具有较小的影响。重组PPV NIa蛋白酶对自裂解具有抗性至少5天。
Site-specific proteases are the most popular kind of enzymes for removing the fusion tags from fused target proteins. Nuclear inclusion protein a (NIa) proteases obtained from the family Potyviridae have become promising due to their high activities and stringencies of sequences recognition. NIa proteases from tobacco etch virus (TEV) and tomato vein mottling virus (TVMV) have been shown to process recombinant proteins successfully in vitro. In this report, recombinant PPV (plum pox virus) NIa protease was employed to process fusion proteins with artificial cleavage site in vitro. Characteristics such as catalytic ability and affecting factors (salt, temperature, protease inhibitors, detergents, and denaturing reagents) were investigated. Recombinant PPV NIa protease expressed and purified from Escherichia coli demonstrated efficient and specific processing of recombinant GFP and SARS-CoV nucleocapsid protein, with site F (N V V V H Q▾A) for PPV NIa protease artificially inserted between the fusion tags and the target proteins. Its catalytic capability is similar to those of TVMV and TEV NIa protease. Recombinant PPV NIa protease reached its maximal proteolytic activity at approximately 30 °C. Salt concentration and only one of the tested protease inhibitors had minor influences on the proteolytic activity of PPV NIa protease. Recombinant PPV NIa protease was resistant to self-lysis for at least five days.
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