title: Production of recombinant Chikungunya virus envelope 2 protein in Escherichia coli.
abstract: Chikungunya, a mosquito-borne viral disease caused by Chikungunya virus (CHIKV), has drawn substantial attention after its reemergence causing massive outbreaks in tropical regions of Asia and Africa. The recombinant envelope 2 (rE2) protein of CHIKV is a potential diagnostic as well as vaccine candidate. Development of cost-effective cultivation media and appropriate culture conditions are generally favorable for large-scale production of recombinant proteins in Escherichia coli. The effects of medium composition and cultivation conditions on the production of recombinant Chikungunya virus E2 (rCHIKV E2) 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 of E. coli expressing rE2 protein. Expression of rCHIKV E2 protein in E. coli was induced with 1 mM isopropyl-beta-thiogalactoside (IPTG) at ~23 g dry cell weight (DCW) per liter of culture and yielded an insoluble protein aggregating to form inclusion bodies. The final DCW after fed-batch cultivation was ~35 g/l. The inclusion bodies were isolated, solubilized in 8 M urea and purified through affinity chromatography to give a final product yield of ~190 mg/l. The reactivity of purified E2 protein was confirmed by Western blotting and enzyme-linked immunosorbent assay. These results show that rE2 protein of CHIKV may be used as a diagnostic reagent or for further prophylactic studies. This approach of producing rE2 protein in E. coli with high yield may also offer a promising method for production of other viral recombinant proteins.
authors: Bioprocess Scale up Facility, Defence Research and Development Establishment, Jhansi Road, Gwalior, 474002, India, tripathink@gmail.com.
authors: Nagesh K
authors: NK
authors: Tripathi
authors: Raj
authors: R
authors: Priya
authors: Ambuj
authors: A
authors: Shrivastava
mesh_terms: D002646:Chikungunya virus / Q000235:genetics*; D002846:Chromatography, Affinity; D003470:Culture Media / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D002479:Inclusion Bodies; D007544:Isopropyl Thiogalactoside / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000378:metabolism*
chemical_list: D003470:Culture Media; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D007544:Isopropyl Thiogalactoside