Jaiswal Smita, Khanna Navin, Swaminathan Sathyamangalam
RGP Laboratory, International Centre for Genetic Engineering and Biotechnology, PO Box 10504, Aruna Asaf Ali Marg, New Delhi 110067, India.
Protein Expr Purif. 2004 Jan;33(1):80-91. doi: 10.1016/j.pep.2003.09.009.
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 approximately 30 mg/L. The purified protein was recognized in Western analyses by monoclonal antibodies specific for the 6x His tag as well as the 3H5 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.
登革病毒感染对全球公共卫生构成严重威胁，目前尚无治疗方法或获批疫苗。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。我们在大肠杆菌中表达并纯化了登革病毒2型包膜的重组结构域III，无需载体蛋白的帮助。在N端插入了一个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。我们通过Ni-NTA亲和层析将重组结构域III蛋白纯化至接近均一性，产量约为30 mg/L。纯化的蛋白在Western分析中被针对6x His标签以及已知位于结构域III中的3H5中和表位的单克隆抗体识别。重组蛋白的真实性也在设计用于特异性同时鉴定6x His标签和3H5表位的夹心ELISA中得到验证。此外，鼠源和人源多克隆血清也识别重组蛋白。体外重折叠的重组蛋白制剂具有生物学功能。它可以有效保护培养中的细胞免受登革病毒2型感染，显然是通过阻止病毒与宿主细胞结合。这种表达/纯化策略具有廉价扩大规模的潜力，可能对登革热诊断和疫苗开发工作有用。
