Recombinant RBD and ACE2 Protein Production

The genes encoding the SARS-CoV-2 S protein RBD (GenBank: QHR63250.2, residues 319–541), SARS-CoV S protein RBD (GenBank: AAP30030.1, residues 306–527), and MERS-CoV S protein RBD (GenBank: AFS88936.1, residues 377–588) were constructed in the expression vector pCAGGS. The chimeric RBDs were also constructed as recently reported^{5 (link)}. The constructs contained a signal peptide at the N-terminus and an IgG1 Fc fragment at the C-terminus. In addition, a thrombin site was inserted between the RBD and Fc fragments. If there is no special indication, RBD indicates the SARS-CoV-2 RBD. Using the same strategy, the encoding recombinant hACE2 (GenBank: NP068576.1, residues 19–615), hACE2-Fc, was also constructed for expression. All proteins were prepared as previously described^{46 (link)}. Briefly, the plasmids were transfected into FreeStyle 293 cells (293F). After five days, the supernatant was collected and purified with Protein A resin (GE Healthcare). The purified proteins were concentrated with a 10-kDa centrifugal filter device (Millipore), and the concentrations were measured by a NanoPhotometer N60 (Implen) with the corresponding extinction coefficient. In addition, part of the purified RBD-Fc protein of SARS-CoV-2 was further digested by thrombin (Sigma-Aldrich) to obtain isolated RBD without the Fc fragment.