Generation and characterization of proteolytically active and highly stable truncated and full-length recombinant West Nile virus NS3
Generation and characterization of proteolytically active and highly stable truncated and full-length recombinant West Nile virus NS3
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DOI：
10.1016/j.pep.2006.10.022
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发表时间：
2007-05-01
影响因子：
1.6
通讯作者：
Young, Paul R.
中科院分区：
文献类型：
--
作者：
Chappell, Keith J.;Stoermer, Martin J.;Young, Paul R.
西尼罗河病毒是一种具有医学意义的新兴病原体，目前尚无有效的抗病毒疗法。 NS2B 和 NS3 编码的病毒蛋白酶是抑制剂开发的一个有吸引力的靶点，并且一直是众多研究的焦点。大多数人都采用了基于我们开发的表达策略的重组蛋白酶，该表达策略通过灵活的甘氨酸接头将 NS2B 内的必需亲水辅因子结构域连接到 NS3 蛋白酶结构域。然而，自蛋白水解一直是与该构建体相关的一个重要问题。例如，最近解析的辅因子结合 WNV NS3 蛋白酶的晶体结构被发现在其 N 末端被截短了 18 个残基。在这项研究中，鉴定了自催化裂解位点，并将其与甘氨酸接头和辅因子结构域的非必需区域一起去除。此外，还确定了 NS3 蛋白酶的最佳大小。基于此优化构建体，掺入全长 NS3 的重组蛋白酶也成功表达和纯化。有点令人惊讶的是，对该重组体的蛋白水解活性与单独的蛋白酶结构域的蛋白水解活性的比较分析表明，NS3 的 C 末端三分之二对底物结合几乎没有影响。这些修饰产生了高度稳定和受限的重组蛋白酶，它们比现有的构建体更适合活性和结构研究。 (c) 2006 Elsevier Inc. 保留所有权利。
West Nile virus is a medically significant emerging pathogen for which there is no effective antiviral therapy. The viral protease encoded by NS2B and NS3 is an attractive target for development of an inhibitor and has been the focus of numerous studies. Most have employed recombinant proteases based on an expression strategy we developed which links the essential hydrophilic cofactor domain within NS2B to the NS3 protease domain by a flexible glycine linker. However, autoproteolysis has been a significant problem associated with this construct. The recently resolved crystal structure of the cofactor bound WNV NS3 protease for example, was found to be truncated by 18 residues at its N-terminus. In this study, the autocatalytic cleavage site was identified and removed along with nonessential regions of the glycine linker and cofactor domain. In addition, the optimal size of the NS3 protease was defined. Based on this optimized construct, a recombinant protease incorporating the full length of NS3 was also successfully expressed and purified. Somewhat surprisingly, comparative analysis of the proteolytic activity of this recombinant with that of the protease domain alone revealed little influence of the C-terminal two thirds of NS3 on substrate binding. These modifications have yielded highly stable and constrained recombinant proteases, which are more suitable than existing constructs for both activity and structural studies. (c) 2006 Elsevier Inc. All rights reserved.