Production and Purification of SARS-CoV-2 Spike Protein

Proteins were produced in Expi293F Cells (ThermoFisher Scientific) grown in suspension using Expi293 Expression Medium (ThermoFisher Scientific) at 37°C in a humidified 8% CO2 incubator rotating at 130 rpm. Cells grown to a density of 3 million cells per mL were transfected using the ExpiFectamine 293 Transfection Kit (ThermoFisher Scientific) and cultivated for 3–5 days. Proteins were purified from clarified supernatants using a nickel HisTrap HP affinity column (Cytiva) and washed with ten column volumes of 20 mM imidazole, 25 mM sodium phosphate pH 8.0, and 300 mM NaCl before elution on a gradient to 500 mM imidazole. To produce SARS-CoV-2 S in the postfusion state, SARS-CoV-2 S D614G was incubated with 1:1 w/w S2X58-Fab (14 (link)) and 10 ug/mL trypsin for one hour at 37C before size exclusion on a Superose 6 Increase column (Cytivia). Proteins were buffer exchanged into 20 mM sodium phosphate pH 8 and 100 mM NaCl and concentrated using centrifugal filters (Amicon Ultra) before being flash frozen.