SARS-CoV-2 Spike Protein S2 Subunit: Recombinant Protein Expression Analysis, Purification, and Its Regulatory Effect on IGF-1R Expression

Abstract

SARS-CoV-2 consists of the spike (S) protein which plays an important role in mediating the entry of virus into the host and it mainly consists of two subunits which are functionally different from each other. The first subunit S1, is involved in binding to the host receptor such as ACE2, and the second subunit S2, is involved in facilitating the viral membrane fusion with that of host membrane. Despite extensive research on the S1 domain due to its immunodominance and variability, the structurally conserved S2 domain remains relatively understudied. Recognizing the potential of S2 in modulating host responses, this study focuses on its recombinant expression, purification, and functional impact. The S2 coding region was cloned into the pGEX2TK plasmid to produce protein, which is a GST fusion-based protein and thereafter, the expression was done in BL21(DE3) strain of Escherichia coli cells. To enhance yield as well as solubility, protein expression was induced at a reduced temperature of 16 °C, minimizing aggregation and degradation. The fusion protein was purified via glutathione affinity chromatography, yielding high-purity S2 suitable for downstream applications. The S2 protein upon transfection into HEK293 and WI-38 mammalian cells leads to the expression of downregulated insulin-like growth factor 1 receptor (IGF-1R), as measured with the help of protein analysis. In order to highlight the role in pathogenesis of COVID-19 through modulation of cellular receptor and for the intervention of therapeutics, S2 can likely be considered a potential target.