Development of a pilot-scale production process and characterization of a recombinant Japanese encephalitis virus envelope domain III protein expressed in Escherichia coli.

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.

Production of recombinant nonstructural 1 protein in Escherichia coli for early detection of Japanese encephalitis virus infection.

Japanese encephalitis is a major public health problem in South-East Asia and Western Pacific countries. The recombinant nonstructural 1 (rNS1) protein of Japanese encephalitis virus is a potential diagnostic as well as vaccine candidate. Developments of cost-effective and simple culture media as well as appropriate culture conditions are generally favourable for large-scale production of recombinant proteins. The effects of medium composition and cultivation conditions on the production of rNS1 protein were investigated in shake flask culture as well as batch cultivation of Escherichia coli. Further, the fed-batch process was also carried out for high cell density cultivation (HCDC) of E. coli expressing rNS1 protein. Isopropyl-ß-d-thiogalactopyranoside (IPTG) was used to induce the expression of rNS1 protein at ∼ 13 g dry cell weight per litre of culture. The final dry cell weight after fed-batch cultivation was ∼ 17 g l(-1) . The Inclusion bodies were isolated and purified through affinity chromatography to give a final product yield of ∼ 142 mg l(-1) . The reactivity of purified protein was confirmed by Western blotting and Enzyme linked immunosorbent assay. These results show that rNS1 protein may be used as a diagnostic reagent or for further prophylactic studies. This approach of producing rNS1 protein in E. coli with high yield may also offer promising method for production of other viral recombinant proteins.

Recombinant dengue virus type 3 envelope domain III protein from Escherichia coli.

Dengue is a public health problem of global significance for which there is neither an effective antiviral therapy nor a preventive vaccine. The envelope protein of dengue virus is the major antigen to elicit neutralizing antibody response and protective immunity in hosts. Optimization of culture media was carried out for enhanced production of recombinant dengue virus type 3 envelope domain III (rDen 3 EDIII) protein in E. coli. Further, batch and fed-batch cultivation process were also developed in optimized medium. After fed-batch cultivation, the dry cell weight was about 22.80 g/L of culture. The rDen 3 EDIII protein was purified using immobilized metal affinity chromatography. This process produced ∼649 mg of purified rDen 3 EDIII protein per liter of culture. The purity of the protein was determined by SDS-PAGE analysis and the reactivity was checked by Western blotting as well as ELISA. These results show that the purified protein may be used for the dengue diagnosis or further prophylactic studies for dengue infection.

Development of a simple fed-batch process for the high-yield production of recombinant Japanese encephalitis virus protein.

Japanese encephalitis (JE) is one of the leading causes of acute encephalopathy affecting children and adolescents in the tropics. Optimization of media was carried out for enhanced production of recombinant JE virus envelope domain III (EDIII) protein in Escherichia coli. Furthermore, batch and fed-batch cultivation process in E. coli was also developed in optimized medium. Expression of this protein in E. coli was induced with 1 mM isopropyl-beta-thiogalactoside and yielded an insoluble protein aggregating to form inclusion bodies. The inclusion bodies were solubilized in 8 M urea, and the protein was purified under denaturing conditions using Ni-NTA affinity chromatography. After fed-batch cultivation, the recombinant E. coli resulted in cell dry weight and purified protein about 36.45 g l(-1) and 720 mg l(-1) of culture, respectively. The purity of the recombinant JE virus EDIII protein was checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, and reactivity of this protein was determined by Western blotting and ELISA with JE virus-infected human serum samples. These results establish the application of this protein to be used for the diagnosis of JE virus infection or for further studies in vaccine development. This process may also be suitable for the high-yield production of other recombinant viral proteins.