Expression, solubilisation, purification and characterisation of recombinant bluetongue virus viral protein 7

Bluetongue virus belongs to the Orbivirus genus from the Reoviridae family. It infects predominantly domestic and wild ruminants and is economically significant worldwide. Bluetongue virus VP7 forms the intercepting layer between the outer capsid (VP2 and VP5) and VP3 which surrounds the genomic material. BL21(DE3), NiCo21(DE3), C43(DE3) pLysS and KRX Escherichia coli cells were transformed with a pET28a plasmid with the cDNA sequence encoding Bluetongue virus VP7. Expression of Bluetongue virus VP7 was tested at post induction temperatures between 16˚C and 37 ˚C, at inducer concentrations between 0.1 mM and 1.0 mM isopropyl-β-D-thiogalactopyranoside in BL21(DE3), NiCo21(DE3) and C43(DE3) pLysS cells and 0.05 % and 0.15 % rhamnose for KRX cells, in two types of growth media (LB and 2xYT) and post-induction growth times between two and 16 hours. Under all conditions tested; Bluetongue virus VP7 expression was found to be predominantly in the insoluble fraction (pellet). BL21(DE3) and NiCo21(DE3) cells were chosen and grown for five hours post induction, induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside and grown at a post-induction temperature of 37 ˚C. Bluetongue virus VP7 in bacterial cell inclusion bodies was solubilised using urea and a freeze-thaw step. Solubilisation was tested with urea concentrations between 2 M and 8 M, with solubilisation efficiency not increasing past 5 M urea. Solubilized Bluetongue virus VP7 was purified using nickel-affinity chromatography. Purified Bluetongue virus VP7 was then probed with far-UV circular dichroism and intrinsic fluorescence in several buffer conditions including different urea and guanidinium chloride concentrations as well as in the presence of glycerol and sodium chloride. Guanidinium chloride was able to cause Bluetongue virus VP7 unfolding, and the unfolding transition had 94 % and 89 % reversibility at 218 nm and 222 nm respectively. Bluetongue virus VP7 was shown to contain a native-like structure in 20 % glycerol and in up to 8 M urea and was found to be stable till at least 55 ˚C, even in the presence of 5 M urea. Glycerol and sodium chloride influenced the conformation of the protein resulting in different unfolding transitions. Thermal unfolding of Bluetongue virus VP7 was found to be irreversible.

Life and Consumer Sciences

M. Sc. (Life Sciences)