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.
Tripathi
Nagesh K
NK
Bioprocess Scale-Up Facility, Defence Research and Development Establishment, Jhansi Road, Gwalior, 474002, India. tripathink@gmail.com
Shukla
Jyoti
J
Biswal
Karttik C
KC
Lakshmana Rao
P V
PV
D000914:Antibodies, Viral / Q000032:analysis / Q000097:blood; D019149:Bioreactors; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D004664:Encephalitis Virus, Japanese / Q000276:immunology; D004672:Encephalitis, Japanese / Q000175:diagnosis; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D006801:Humans; D007075:Immunoglobulin M / Q000032:analysis; D002479:Inclusion Bodies / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
D000914:Antibodies, Viral; D003470:Culture Media; D007075:Immunoglobulin M; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins