Engineering rotavirus viral protein 6 consensus sequence to reduce aggregation for expression and purification

Abstract

The VP6 protein sequence from Rotavirus A was analysed using AGGRESCAN3D to identifying aggregation-prone regions. Six amino acid residues (four Valine, one Phenylalanine, and one Isoleucine) were mutated to reduce aggregation. VP6 consensus (wild-type) and VP6 mutant genes were cloned into pET-15b for expression with BL21(DE3) and NiCo21(DE3) using different conditions. NiCo21(DE3) yielded the highest expression in inclusion bodies at 37°C, 0.04 mM IPTG, after 12 hours. An optimized wash step was added to improve purity, and optimal solubilisation was achieved with 7 M urea for VP6 wild-type protein, while the VP6 mutant protein required 5 M and 7 M urea, demonstrating significantly improved solubility, following a freeze-thaw cycle. Purification by Immobilized Metal Affinity Chromatography (IMAC) showed VP6 co-eluting with E. coli proteins confirmed by mass spectrometry: EF-Tu and PheS with VP6 wild-type protein, and YcfT and MalE with VP6 mutant protein. Structural analysis using Far UV Circular Dichroism (CD), Attenuated Total Reflectance–Fourier Transform Infrared Spectroscopy (ATR-FTIR), and fluorescence spectroscopy, showed a complete loss of secondary and tertiary structure at 7 M urea, whereas the VP6 mutant protein showed significant unfolding at 5 M urea, indicating reduced structural stability. Thermal denaturation monitored by Far-UV circular dichroism and intrinsic fluorescence showed consistent unfolding behaviour for VP6 proteins. The VP6 wild-type protein began unfolding at 35°C, while the VP6 mutant protein showed a greater thermal stability, initiating unfolding at 45°C. Despite this difference, neither protein demonstrated refolding upon cooling, indicating irreversible denaturation. Native-PAGE showed VP6 forming higher-order quaternary structures. The VP6 wild-type protein formed multiple oligomeric bands, while the VP6 mutant protein formed fewer, weaker bands, indicating reduced oligomeric complexity and disrupted quaternary stability