Protein a affinity column

RAY-53-IgG (Huang et al. 2021) was produced in ExpiCHO Cells (ThermoFisher Scientific) grown in suspension using ExpiCHO Expression Medium (ThermoFisher Scientific) at 37°C in a humidified 8% CO2 incubator rotating at 130 rpm. Cells grown to a density of 6 million cells per mL were transfected using the ExpiFectamine CHO Transfection Kit (ThermoFisher Scientific) and cultivated for 6–8 days. Proteins were purified from clarified supernatants using a Protein A affinity column (Cytiva) and washed with ten column volumes of 20 mM sodium phosphate pH 8.0 before elution with 0.1 M citric acid pH 3 into 1 M Tris-HCl pH 9. 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.

FreeStyle 293F cells (Invitrogen, Rockford, IL, USA) were grown in FreeStyle 293F medium (Invitrogen) to a density of 1 × 10⁶ cells/mL at 37 °C with 8% CO₂ with regular agitation (150 rpm). Cells were transfected with a plasmid coding for soluble ACE2 or ACE2-Fc using ExpiFectamine 293 transfection reagent, as directed by the manufacturer (Invitrogen). One week later, cells were pelleted and discarded. Supernatants were filtered using a 0.22 µm filter (Thermo Fisher Scientific, Waltham, MA, USA). The recombinant sACE2 protein was purified by nickel affinity columns (Invitrogen) and ACE2-Fc was purified using protein A affinity column (Cytiva, Marlborough, MA, USA), as directed by the manufacturers. The protein preparations were dialyzed against phosphate-buffered saline (PBS) and stored in aliquots at −80 °C until further use. To assess purity, recombinant proteins were loaded on SDS-PAGE gels and stained with Coomassie blue.

FreeStyle 293F cells (Invitrogen, Waltham, MA, USA) were grown in FreeStyle 293F medium (Invitrogen) to a density of 1 × 10⁶ cells/mL at 37 °C with 8% CO₂ with regular agitation (150 rpm). Cells were transfected with a plasmid coding for SARS-CoV-2 Omicron BA.2 S RBD (319-537), soluble ACE2 (sACE2, 1-615), or ACE2-Fc (1-615), using ExpiFectamine 293 transfection reagent, as directed by the manufacturer (Invitrogen). One week later, cells were pelleted and discarded. Supernatants were filtered using a 0.22 µm filter (Thermo Fisher Scientific, Waltham, MA, USA). The recombinant sACE2 protein and RBD proteins were purified by nickel affinity columns, as directed by the manufacturer (Invitrogen) and ACE2-Fc was purified using a protein A affinity column (Cytiva, Marlborough, MA, USA), as directed by the manufacturer. The protein preparations were dialysed against phosphate-buffered saline (PBS) and stored at −80 °C in aliquots until further use. To assess purity, recombinant proteins were loaded on SDS-PAGE gels and stained with Coomassie Blue. Purified proteins were >95% pure after size-exclusion chromatography as verified by SDS-PAGE and Coomassie blue staining.