Expression and Purification of SARS-CoV-2 Spike and ACE2

The SARS-CoV-2 RBD (residues Arg319–Phe541) and the N-terminal peptidase domain of human ACE2 (residues Ser19–Asp615) were expressed using the Bac-to-Bac baculovirus system (Invitrogen) as described previously[21 (link)]. ACE2-lg, a recombinant Fc fusion protein of soluble human ACE2 (residues Gln18-Ser740), was expressed in 293F cells and purified using protein A affinity chromatography as described in our previous study[36 (link)]. The cDNA of extracellular domain (ECD) (1–1208 aa) of WT or mutant spike proteins of SARS-CoV-2 were cloned into the pCAG vector with a C-terminal T4 fibritin trimerization motif followed by a 8✕his tag and two strep tag. The recombinant protein was overexpressed using the HEK293F mammalian cells 5 days after transfection. The secreted proteins were purified by streptavidin affinity resin and size-exclusion chromatography in buffer containing 25 mM Tris (pH 8.0), 150 mM NaCl.