To express SARS-CoV-2 S glycoprotein ectodomain, the mammalian codon-optimized gene coding SARS-CoV-2 (Wuhan-Hu-1 strain, GenBank ID: MN908947.3) S glycoprotein ectodomain (residues M1–Q1208) with proline substitutions at K986 and V987, a “GSAS” substitution at the furin cleavage site (R682 to R685) was cloned into vector pcDNA 3.1+. A C-terminal T4 fibritin trimerization motif, a TEV protease cleavage site, a FLAG tag, and a His tag were cloned downstream of the SARS-CoV-2 S glycoprotein ectodomain (fig. S1A). A gene encoding human ACE2 PD domain (Q18-D615) with an N-terminal interleukin-10 (IL-10) signal peptide and a C-terminal His tag was cloned into vector pcDNA 3.4. The expression vectors were transiently transfected into HEK293F cells using polyethylenimine. Three days after transfection, the supernatants were harvested. To purify the His-tagged S and ACE2 proteins, the clarified supernatants were added with 20 mM tris-HCl (pH 7.5), 200 mM NaCl, 20 mM imidazole, and 4 mM MgCl2, and incubated with Ni–nitrilotriacetic acid (NTA) resin at 4°C for 1 hour. The Ni-NTA resin was recovered and washed with 20 mM tris-HCl (pH 7.5), 200 mM NaCl, and 20 mM imidazole. The protein was eluted by 20 mM tris-HCl (pH 7.5), 200 mM NaCl, and 250 mM imidazole.

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