High-level expression and one-step purification of recombinant dengue virus type 2 envelope domain III protein in Escherichia coli
High-level expression and one-step purification of recombinant dengue virus type 2 envelope domain III protein in Escherichia coli
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DOI：
10.1016/j.pep.2003.09.009
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发表时间：
2004-01-01
影响因子：
1.6
通讯作者：
Swaminathan, S
中科院分区：
文献类型：
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作者：
Jaiswal, S;Khanna, N;Swaminathan, S
登革病毒感染对全球公共卫生构成严重威胁，目前没有治疗方法或许可的疫苗。登革病毒编码的包膜蛋白的结构域III携带多个构象依赖性中和表位，对病毒感染性至关重要。我们已经表达和纯化的重组结构域III的登革病毒2型包膜，没有一个载体蛋白的帮助下，在大肠杆菌。在N末端插入6x His标签以促进其一步纯化。该蛋白以不溶性包涵体的形式过表达，在高度变性条件下溶解，然后进行先前优化的精氨酸介导的复性方案。用Ni-NTA亲和层析法对重组蛋白进行了纯化，得到了约30 mg/L的纯化产物。纯化的蛋白质在Western分析中被特异于6x His标签以及已知存在于结构域III中的3145中和表位的单克隆抗体识别。重组蛋白的真实性也在夹心ELISA中得到验证，该夹心ELISA设计用于特异性地同时鉴定6x His标签和3 H5表位。此外，鼠和人多克隆血清也识别重组蛋白。体外复性的重组蛋白制剂具有生物学功能。它可以有效地保护培养细胞免受登革病毒2型感染，显然是通过阻止病毒与宿主细胞结合。这种表达/纯化策略具有廉价放大的潜力，并且可能被证明对登革热诊断和疫苗开发工作有用。(C)2003年爱思唯尔公司All rights reserved.
Dengue virus infection poses a serious global public health threat for which there is currently no therapy or a licensed vaccine. The domain III of the dengue virus encoded envelope protein, which carries multiple conformation-dependent neutralizing epitopes, is critical for virus infectivity. We have expressed and purified recombinant domain III of dengue virus type-2 envelope, without the aid of a carrier protein in Escherichia coli. A 6x His tag was inserted at the N terminus to facilitate its one-step purification. The protein was overexpressed in the form of insoluble inclusion bodies, which were solubilized under highly denaturing conditions and then subjected to a previously optimized arginine-mediated renaturation protocol. We purified recombinant domain III protein to near homogeneity by Ni-NTA affinity chromatography and obtained yields of similar to30mg/L. The purified protein was recognized in Western analyses by monoclonal antibodies specific for the 6x His tag as well as the 3145 neutralizing epitope known to reside in domain III. The authenticity of the recombinant protein was also verified in a sandwich ELISA designed to specifically and simultaneously identify the 6x His tag and the 3H5 epitope. In addition, murine and human polyclonal sera also recognized the recombinant protein. The in vitro refolded recombinant protein preparation was biologically functional. It could effectively protect cells in culture against dengue virus type-2 infection, apparently by blocking the virus from binding to host cells. This expression/ purification strategy has the potential for inexpensive scale-up and may prove to be useful for dengue diagnostics and vaccine development efforts. (C) 2003 Elsevier Inc. All rights reserved.