Pilot-scale characterization and purification of recombinant tagged Chikungunya virus structural proteins.
Chikungunya virus (CHIKV) is swiftly emerging as a wide-reaching health menace. In this study, the sequences of recombinant tagged CHIKV structural proteins E3, E2, E1 and 6K were analysed to predict potential protein features and possible antigenic sites. All four recombinant proteins were inducibl...
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| 格式: | Final Year Project |
| 語言: | English |
| 出版: |
2009
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| 在線閱讀: | http://hdl.handle.net/10356/16355 |
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| 機構: | Nanyang Technological University |
| 語言: | English |
| 總結: | Chikungunya virus (CHIKV) is swiftly emerging as a wide-reaching health menace. In this study, the sequences of recombinant tagged CHIKV structural proteins E3, E2, E1 and 6K were analysed to predict potential protein features and possible antigenic sites. All four recombinant proteins were inducibly expressed in an E. coli expression system and characterised in terms of solubility in Tris-HCl/NaCl (TN) buffer, 8 M urea denaturing buffer and 6 M guanidine hydrochloride (Gn-HCl) denaturing buffer. All four recombinant proteins were found to be insoluble in TN buffer. Recombinant E3, E2 and 6K were partially soluble in 8 M urea denaturing buffer, but E1 was almost completely insoluble. 6 M Gn-HCl denaturing buffer successfully solubilised all four recombinant proteins. 6 M Gn-HCl extracts of the recombinant proteins were purified by binding to metal-affinity resins by means of the 6-histidine (6xHis) tag epitope, and elutions of the recombinant proteins obtained. Future progress will involve the scale-up of the process used here to produce purified recombinant proteins for raising monoclonal antibodies. Based on prior characterization, E1 and E2 are thus expected to be particularly suited for use as antigenic targets for raising monoclonal antibodies. |
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