Purification of SARS-CoV-2 RBD Proteins

Escherichia coli cells carrying the recombinant plasmids encoding for CΔ116-MrNV-CP^β-RBD or CΔ116-MrNV-CP^δ-RBD were inoculated into 50 mL LB broth containing ampicillin (100 μg/mL) and cultured overnight at 37 °C. An aliquot (10 mL) of overnight culture was transferred to 1 L LB broth and incubated at 37 °C until the culture reached an OD₆₀₀ of ~0.6. Protein expressions were induced by adding 1 mM IPTG into the culture, and it was incubated at 25 °C for 5 h. The cell pellets were harvested by centrifugation at 8000× g for 8 min. Purification of recombinant proteins was modified from a previous study by Kumar et al. [21 (link)], using a fast-protein liquid chromatography (FPLC) system (Äkta Purifier; GE Healthcare, Uppsala, Sweden). The HiTap^TM SP HP 1 mL column (GE Healthcare, Buckinghamshire, UK) was washed with 10 column volume (CV) of binding buffer (50 mM HEPES, 100 mM NaCl, pH 7.4). The lysates were loaded onto the column, and bound proteins were eluted via a gradient of NaCl concentration (50 mM HEPES, 1 M NaCl, pH 7.4) at a flow rate of 1 mL/min and fractionated using a Frac-950 collector. The eluted proteins were then analyzed with SDS-PAGE and Western blotting.