Expression, purification, and characterisation of the Alpha-helical and Beta-sheet domains of Rotavirus VP6

Abstract

The capsid protein VP6 is of paramount importance to the stability and infectivity of Rotaviruses. Through interactions of VP6s’ beta-sheet (VP6) and alpha-helical (VP6) domains with the viral particle's outer- and innermost layers, respectively, VP6 stabilises matured Rotaviruses and activates transcription of the viral genome upon cell entry. This study focused on the individual domains of Rotavirus VP6. The aim of the study was to probe the structure and stability of VP6 and VP6 when expressed independently of each other. The objectives of the study were: (1) optimise the bacterial expression of VP6 and VP6 through modulation of the expression conditions (2) solubilise and then purify the domains by immobilised metal chromatography (IMAC), (3) characterise by means of spectroscopy (mass spectroscopy, far-UV circular dichroism (CD), and intrinsic tryptophan fluorescence spectroscopy) and gel electrophoresis (native-PAGE), the primary, secondary, tertiary, and quaternary structures of the domains, (4) characterise the conformational stability by means of spectroscopy (far-UV CD and intrinsic tryptophan fluorescence) of VP6 and VP6 when thermally and chemically challenged, and (5) determine the melting temperature by differential scanning calorimetry (DSC). To this end, two Escherichia coli strains BL21(DE3) and NiCo21 (DE3) were transformed with pET15a plasmids containing the codon-optimised DNA consensus sequences of VP6 and VP6. The expression of VP6 and VP6 was done at different temperatures (37°C and 20°C), inducer concentrations (1 × and 10 × IPTG), and post-induction incubation times (2 h – 7 h, and 16 h) in both E. coli strains and the outcomes were visualised by SDS-PAGE. All conditions tested produced the domains in an insoluble form and though expression levels appeared to be comparable between strains, the NiCo21 (DE3) was ultimately selected for further expression of the domains as expression could be induced with the lowest concentration of IPTG. The insoluble domains were subjected to a solubilisation study where the domains were frozen in various Tris-HCl buffers differing in pH (7 – 10) and urea concentration (0 M, 2 M, and 5 M) and thawed. The results of the solubilisation study showed that both domains could effectively be solubilized in 2 M urea, provided that the pH of the freezing buffer was at least one unit higher than the pI of the domain. The solubilised VP6 and VP6 were purified by nickel affinity chromatography in yields of 13.32 mg and 25 mg from 1 L of NiCo21 (DE3) culture, respectively and were confirmed by mass spectroscopy, far-UV CD, and intrinsic tryptophan fluorescence spectroscopy, to have native-like sequences and structural features. The quaternary analysis revealed that VP6 existed as a single monomeric species in solution while VP6 formed different-sized structures in solution. The conformational stability of VP6 and VP6 was demonstrated as the domains had resisted structural changes up to 46°C and 50°C, respectively and the DSC analysis revealed melting points of 67.94°C for VP6 and 68.55°C for VP6. The domains were noted to aggregate extensively which prevented the recovery of their native structures upon cooling. The chemical unfolding study was done in 1 M – 5 M guanidine hydrochloride (GdCl) and 1 M – 8 M urea, and revealed the chemical stability of the domains and their respective unfolding pathways. Approximately 1.5 M and 2.25 M GdCl were needed to denature 50% of VP6 and VP6, respectively. Urea concentrations of 4.5 M (VP6) and 4 M (VP6) also resulted in a 50% loss of native structures. The observation of non-cooperative unfolding pathways that differed between the spectroscopic probes suggested a complex unfolding process involving the formation of one or more intermediates. Though native-like structures could be recovered upon denaturant removal, the refolding and unfolding pathways differed, which was indicative of irreversibility. Overall, VP6 and VP6 were easily producible and purifiable in quantities suitable for further studies. Further investigations could highlight the potential applications the domains could have in vaccine development and drug-delivery. Non-cooperative folding indicated the necessity of interactions between VP6 and VP6 in the full-length protein for cooperative folding.