Optimization of purification and characterisation of over-expressed rotavirus capsid protein VP6
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Authors
Kgokolo, Samuel Maphalle
Issue Date
2017-12
Type
Dissertation
Language
en
Keywords
Chromatography , Circular dichroism , Escherichia coli , Fluorescence , Plasmid , Protein conformation , Protein expression , Purification , Rotavirus , VP6
Alternative Title
Abstract
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.
Description
Citation
Kgokolo, Samuel Maphalle (2017) Optimization of purification and characterisation of over-expressed rotavirus capsid protein VP6, University of South Africa, Pretoria, <http://hdl.handle.net/10500/23726>