Recombinant Human ACE2 Extracellular Domain Production

The extracellular domain of ACE2 receptor (GenBank NM_021804.3) was produced in pCSE2.6-hFc expression vector in Expi293F cells (Thermo Fisher Scientific) as described before [22 (link)]. In brief, Expi293F cells were cultivated at 37°C, 110 rpm, and 5% CO₂ in Gibco FreeStyle F17 expression media (Thermo Fisher Scientific) supplemented with 8 mM Glutamine and 0.1% Pluronic F68 (PAN Biotech). For transfection, 1 μg DNA and 5 μg of 40 kDa PEI (Polysciences) per mL transfection volume were diluted separately in 5 transfection volumes and then mixed for the formation of complexes (20–30 min). Afterwards, PEI:DNA complexes were added to 1.5–2 × 10⁶ cells/mL. Forty-eight hours later, the culture volume was doubled by feeding HyClone SFM4Transfx-293 media (GE Healthcare) supplemented with 8 mM Glutamine and HyClone Boost 6 supplement (GE Healthcare) with 10% of the end volume. One week after transfection, the supernatant was harvested by 15 min centrifugation at 1500 ×g. Purification was performed on a 1-mL HiTrap Fibro PrismA (Cytiva) column on Äkta go (Cytiva) according to the manufacturer’s manual.