Centrifugal ultrafiltration devices

Plasmids (500 ng DNA per ml culture) and polyethylenimine (MW 25,000; Polysciences; 3 μg per ml culture) were mixed with OptiMEM (Gibco; 100 μl per ml culture), incubated 20 minutes at room temperature and added to Expi293F cells at a density of 2 × 10⁶ / ml. Transfection Enhancers (ThermoFisher) were added 18–23 h post-transfection. Culture supernatant was harvested 4–6 days later by two centrifugation steps (800 × g for 10 minutes to remove cells and 20,000 × g for 20 minutes to remove debris). IgG1 Fc fused and 8his-tagged proteins were subsequently purified as previously described (27 ) using KANEKA KanCapA 3G Affinity (Pall) and HisPur Ni-NTA (Thermo Scientific) resins, respectively. Eluted proteins from affinity chromatography were then separated on a Superdex 200 Increase 10/300 GL column (GE Healthcare Life Sciences) equilibrated with Dulbecco’s phosphate-buffered saline (PBS). Proteins from peak fractions were concentrated using centrifugal ultrafiltration devices (Millipore) to final concentrations of ~1 mg/ml (RBD-8h proteins), ~10 mg/ml (sACE2₂-8h proteins) and ~50 mg/ml (sACE2₂-IgG1 proteins). Concentrations were determined by absorbance at 280 nm using calculated extinction coefficients. Reported concentrations for sACE2₂ are based on monomeric subunits. Aliquots were snap frozen in liquid N₂ and stored at −80 °C.