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1	Optimization of purification and characterisation of over-expressed rotavirus capsid protein VP6 Kgokolo, Samuel Maphalle 12 1900 (has links) Rotavirus is responsible for the death of many children annually, and current vaccines have lower efficiency in developing countries. A reverse translated consensus gene sequence of the rotavirus VP6 cloned into a pET-28a(+) plasmid was used to transform BL21 and KRX Escherichia coli cells. Optimal expression of soluble protein was induced in KRX cells by adding 0.05% L-rhamnose and 0.0001 M IPTG, with an incubation temperature of 25ºC for 6 h. VP6 was purified by combining anion exchange chromatography followed by affinity chromatography. Far-UV circular dichroism and intrinsic fluorescence were used as probes to assess the native structure of VP6 and structural in the presence of a denaturant, high sodium chloride concentrations and varying temperatures. The 0.2 M sodium chloride had an impact on the VP6’s tertiary structure and also influenced the proteins conformational changes as detected during thermal unfolding to 90ºC. Although treatment with 3 M urea showed tertiary structural changes no secondary structural loss occurred due to the presence of a denaturant. / Life Sciences / M. Sc. (Life Sciences) Chromatography Circular dichroism Escherichia coli Fluorescence Plasmid Protein conformation Protein expression Purification Rotavirus VP6 616.926 Rotavirus infections Molecular epidemiology Chromatographic analysis Gastritis -- Microbiology Escherichia coli infections

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Optimization of purification and characterisation of over-expressed rotavirus capsid protein VP6