title: Development of a pilot-scale production process and characterization of a recombinant Japanese encephalitis virus envelope domain III protein expressed in Escherichia coli.
abstract: Japanese encephalitis virus (JEV) is the most important cause of encephalitis in most Asian regions. JEV envelope domain III (JEV EDIII) protein is involved in binding to host receptors, and it contains specific epitopes that elicit virus-neutralizing antibodies. A highly immunogenic, recombinant JEV EDIII protein was expressed in Escherichia coli. In order to take this vaccine candidate for further studies, recombinant JEV EDIII protein was produced employing a pilot-scale fermentation process. Recombinant JEV EDIII protein expressed as inclusion bodies (IBs) was solubilized in 8 M urea and renatured by on-column refolding protocol in the presence of glycerol. A three-step purification process comprising of affinity chromatography, ion-exchange chromatography (IEX) based on salt, and IEX based on pH was developed. About ~124 mg of highly purified and biologically active EDIII protein was obtained from 100 g of biomass. Biological function of the purified EDIII protein was confirmed by their ability to generate EDIII-specific antibodies in mice that could neutralize the virus. These findings suggest that recombinant JEV EDIII protein in combination with compatible adjuvant is highly immunogenic and elicit high-titer neutralizing antibodies. Thus, recombinant JEV EDIII protein produced at large scale can be a potential vaccine candidate.
authors.affiliation: Bioprocess Scale Up Facility, Defence Research and Development Establishment, Jhansi Road, Gwalior, 474002, India. tripathink@gmail.com
authors.forename: Nagesh K
authors.initials: NK
authors.lastname: Tripathi
authors.forename: Ambuj
authors.initials: A
authors.lastname: Shrivastava
authors.forename: Karttik C
authors.initials: KC
authors.lastname: Biswal
authors.forename: P V Lakshmana
authors.initials: PV
authors.lastname: Rao
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000097:blood; D000914:Antibodies, Viral / Q000097:blood; D001709:Biotechnology / Q000379:methods; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004664:Encephalitis Virus, Japanese / Q000235:genetics / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics*; D005285:Fermentation; D015870:Gene Expression; D051379:Mice; D009500:Neutralization Tests; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D022223:Vaccines, Subunit / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D014614:Vaccines, Synthetic / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D014765:Viral Vaccines / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D011994:Recombinant Proteins; D022223:Vaccines, Subunit; D014614:Vaccines, Synthetic; D014759:Viral Envelope Proteins; D014765:Viral Vaccines