title: High-level expression and one-step purification of recombinant dengue virus type 2 envelope domain III protein in Escherichia coli.
abstract: 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.
authors.affiliation: RGP Laboratory, International Centre for Genetic Engineering and Biotechnology, PO Box 10504, Aruna Asaf Ali Marg, New Delhi 110067, India.
authors.forename: Smita
authors.initials: S
authors.lastname: Jaiswal
authors.forename: Navin
authors.initials: N
authors.lastname: Khanna
authors.forename: Sathyamangalam
authors.initials: S
authors.lastname: Swaminathan
mesh_terms: D000818:Animals; D001120:Arginine / Q000737:chemistry; D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods; D006224:Cricetinae; D003716:Dengue Virus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D019791:Guanidine / Q000737:chemistry; D010957:Plasmids / Q000235:genetics; D020673:Protein Renaturation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D010948:Viral Plaque Assay / Q000379:methods
chemical_list: C051821:E-glycoprotein, Dengue virus type 2; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D001120:Arginine; D019791:Guanidine