Recombinant SARS-CoV-2 Spike Protein Production

Recombinant WA1, Beta, Alpha, and Omicron spike proteins and recombinant WA1, Beta, Alpha, Gamma, Delta, and Omicron RBDs were generated and expressed in Expi293F cells (Life Technologies, Thermo Fisher Scientific) as previously described (26 (link), 27 (link)). Proteins were then purified after transient transfections with each respective plasmid. Briefly, the mammalian-cell codon-optimized nucleotide sequence of a soluble spike protein (amino acids 1–1,213) lacking the polybasic cleavage site, carrying two stabilizing mutations (K986P and V987P), a signal peptide, and at the C-terminus a thrombin cleavage site, a T4 fold-on trimerization domain, and a hexahistidine tag was cloned into the mammalian expression vector pCAGGS (https://www.beiresources.org/). Protein was purified using gravity flow purification with Ni-nitrilotriacetic acid (NTA) agarose (Qiagen, Germany) and concentrated, and buffer exchanged in Amicon centrifugal units (EMD Millipore, MA, USA). The purified recombinant proteins were analyzed via reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The desired protein folding was confirmed through ELISAs using the Receptor Binding Domain (RBD)-specific monoclonal antibody CR3022 (28 (link)). Recombinant Gamma (10795-CV-100) and Delta (10878-CV-100) spike proteins were purchased from R&D Systems (R&D Systems, Bio-Techne, MN, USA).