Recombinant SARS-CoV-2 Spike Protein Purification

The cloning, expression, and purification of the N-terminal region of the Spike protein were previously described [8 (link)]. Methodology details are in a supplementary file (Figure S7). The SARS-CoV-2 Spike DNA fragment corresponding to the residues from 16 to 165 (rSpike) was amplified by PCR using a SARS-CoV-2 cDNA. The purified PCR product was digested using AnzaTM restriction enzymes NheI and BamHI (Thermo Fisher Scientific, Waltham, MA, USA), and the same pair of enzymes were used to digest the expression vector pET-28a. The digested and purified plasmid was used to ligate the rSpike DNA fragment. Digestion tests confirmed the positive clones. This cloning results in a fusion of seven histidine tags at the N-terminal portion of the protein. rSpike was purified by expression of the protein in Escherichia coli strain BL21(DE3) or BL21 StarTM (DE3), followed by two steps of purification using HisTrap Chelating HP column (GE Healthcare Life Sciences, Chicago, IL, USA) and HiLoad 16/600 Superdex 75 pg (GE Healthcare Life Sciences) size exclusion chromatography. The purified protein (7 M urea, 50 mM MOPS, 200 mM NaCl, 1 mM EDTA, pH 7.0) was concentrated using Amicon Ultra-15 Centrifugal filters (Merck Millipore, Milwaukee, WI, USA) with a 3 kDa membrane cut-off.