2.1. Expression and purification of the S glycoprotein and ACE2
SARS-CoV-2 S glycoprotein expression plasmids were constructed to encode the ectodomain of S glycoprotein (residues 1–1208, with a mutated furin cleavage site and two stabilizing proline substitutions K986P/V987P) followed by a T4 foldon trimerization domain and a polyhistidine purification tag, as described previously 34. For functional assays, different constructs were designed to encode S with mutations found in wild-type human viruses or viruses from American mink and are referred to throughout the text as SD614G, SD614G+Y453F, and SD614G+Δ69-70+Y453F. For structural analysis, a SARS-CoV-2 S glycoprotein expression plasmid with four additional stabilizing proline mutations (F817P, A892P, A899P, A942P)35 was constructed and is referred to throughout the text as mink S glycoprotein for simplicity.
ACE2 expression plasmids were also constructed as described previously to encode the ectodomain of dimeric ACE2 followed by a human IgG1 Fc purification tag, referred to throughout the text as hACE2-Fc and mvACE2-Fc 34. Additionally, a monomeric mink ACE2 construct, where the IgG1 Fc purification tag was replaced with a polyhistidine purification tag, was constructed for structure determination and is referred to throughout the text as mvACE2. The American mink ACE2 sequence was determined previously36.
Expi293F cells (Thermo Fisher, A14527) were cultured in Expi293 Expression Medium at 37°C and 8% CO2 in the Orbi-Shaker (Sigma, Z763438) at 130 rpm. The cells were transiently transfected at a 3.0 x 106 cells/mL density using an Expifectamine 293 transfection kit (Thermofisher, A14524). For polyhistidine-tagged proteins, the supernatant was harvested by centrifugation after 72 hours and supplemented with 20 mM Imidazole, followed by affinity chromatography with Ni2+-NTA agarose beads (GoldBio, H-350). The sample was washed three times with 5 column volumes (CV) of wash buffer (30 mM Na2HPO4 pH 7.4, 500 mM NaCl, 30 mM Imidazole) and then eluted three times with 5 CVs of elution buffer (20 mM Na2HPO4 pH 7.4, 500 mM NaCl, 500 mM Imidazole). For Fc-tagged proteins, the supernatant was harvested by centrifugation after 72 hours, followed by affinity chromatography using HiTrap protein A columns (Cytiva). The sample was washed with 10 CVs of wash buffer (20 mM sodium phosphate, pH 7.4) and eluted with 5 CVs of elution buffer (0.1 M citric acid, pH 3). Sample collection tubes were pre-loaded with neutralization buffer (1M Tris HCl, pH 9).
The eluted proteins were concentrated with 50-kDa or 100-kDa MWCO centrifugal filters. Protein samples used for bio-layer interferometry (BLI) assays were used after affinity chromatography. Protein samples used for structure determination were further purified by size exclusion chromatography using the Superose 6 increase 10/300 GL column (Cytiva) in buffer (20 mM Tris pH 8.0, 200 mM NaCl). The peak fractions were collected and verified through SDS-PAGE gel analysis. The samples were concentrated and flash-frozen in liquid nitrogen and stored at -80°C.