Journal Article

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

Biswal, Karttik C, Lakshmana Rao, P. V, Tripathi, Nagesh K, Shukla, Jyoti

2010

Overview

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-β-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.

• Publisher: Berlin/Heidelberg : Springer-Verlag,Springer-Verlag,Springer,Springer Nature B.V
• Subject: adolescents / Affinity chromatography / Analysis / Antibodies / Antibodies, Viral / Antibodies, Viral - analysis / Antibodies, Viral - blood / Antigens / Bacteria / Batch culture / Biological and medical sciences / Biomedical and Life Sciences / Bioreactors / biosynthesis / Biotechnological Products and Process Engineering / Biotechnology / blood / Blotting, Western / Cell culture / chemistry / children / Chromatography, Affinity / Cloning / Cultivation / Culture Media / Dengue fever / Development and progression / Diagnosis / E coli / Electrophoresis / Electrophoresis, Polyacrylamide Gel / Encephalitis / Encephalitis Virus, Japanese / Encephalitis Virus, Japanese - immunology / Encephalitis, Japanese / Encephalitis, Japanese - diagnosis / Encephalopathy / Enzyme-Linked Immunosorbent Assay / Escherichia coli / Escherichia coli - genetics / Escherichia coli - growth & development / Escherichia coli - metabolism / Ethylenediaminetetraacetic acid / Fed batch / Fundamental and applied biological sciences. Psychology / genetics / Glycerol / growth & development / Health aspects / Humans / Immunoglobulin M / Immunoglobulin M - analysis / immunology / Inclusion bodies / Inclusion Bodies - metabolism / isolation & purification / Japanese encephalitis / Japanese encephalitis virus / Laboratories / Life Sciences / Medical research / metabolism / Methods / Microbial Genetics and Genomics / Microbiology / Optimization / Pharmaceuticals / Physiological aspects / Polyacrylamide / polyacrylamide gel electrophoresis / protein synthesis / Proteins / Public health / Recombinant Proteins / Recombinant Proteins - biosynthesis / Recombinant Proteins - chemistry / Recombinant Proteins - immunology / Recombinant Proteins - isolation & purification / Sodium dodecyl sulfate / Sodium lauryl sulfate / Studies / Sulfates / Tropical environments / tropics / Urea / Vaccine development / Vaccines / Vector-borne diseases / Viral Envelope Proteins / Viral Envelope Proteins - biosynthesis / Viral Envelope Proteins - genetics / Viral Envelope Proteins - immunology / Viral Envelope Proteins - isolation & purification / Viral proteins / Viruses / Western blotting / Western immunoblotting