The SARS-CoV-2 RBD WT (Wuhan Hu-1) encoding sequence was codon usage-optimized and synthesized by GeneArt AG (part of Thermo Fisher Scientific Inc.). The RBD was subsequently cloned into a modified pcDNA5/FRT/TO encoding an N-terminal tPA signal peptide and a C-terminal avi-hexahistidine tag (sequence: GGSGLNDIFEAQKIEWHEGSHHHHHH). The plasmid encoding the RBD of SARS-CoV-2 Delta V1 (B.1.617.2) was generated by introducing the corresponding mutations L452R and T478K via overlap extension PCR (Higuchi R et al., 1988 (link)) in the WT sequence and by re-cloning into the original pcDNA5/FRT/TO-derivate.
For expression and purification of the antigens, Expi293F™ cells (Thermo Fisher Scientific; A14527) were transfected with plasmids encoding the SARS-CoV-2 RBD variants according to the manufacturer’s recommendations. After 5 days of protein expression, supernatants were harvested by centrifugation. Supernatants were loaded onto an immobilized metal chelate affinity chromatography column (HisTrap Excel, Cytiva), washed with Dulbecco’s Phosphate Buffered Saline (PBS, Sigma) containing 10 mM imidazole (Sigma) and eluted over a linear gradient of 10–500 mM imidazole in PBS. The protein was buffer exchanged to PBS and concentrated to approximately 1–2 mg/ml by ultrafiltration.
For expression and purification of the antigens, Expi293F™ cells (Thermo Fisher Scientific; A14527) were transfected with plasmids encoding the SARS-CoV-2 RBD variants according to the manufacturer’s recommendations. After 5 days of protein expression, supernatants were harvested by centrifugation. Supernatants were loaded onto an immobilized metal chelate affinity chromatography column (HisTrap Excel, Cytiva), washed with Dulbecco’s Phosphate Buffered Saline (PBS, Sigma) containing 10 mM imidazole (Sigma) and eluted over a linear gradient of 10–500 mM imidazole in PBS. The protein was buffer exchanged to PBS and concentrated to approximately 1–2 mg/ml by ultrafiltration.