Expression of SARS-CoV-2 spike recombinant protein

Mammalian cell codon-optimized nucleic sequence, coding for SARS-CoV-2 spike glycoprotein (GenPept: {"type":"entrez-protein","attrs":{"text":"QHD43416","term_id":"1791269090","term_text":"QHD43416"}}QHD43416 ORF [https://www.ncbi.nlm.nih.gov/protein/1791269090]), was used to design pcDNA3.1+ based expression plasmids, mediating recombinant expression of the entire spike glycoprotein (amino acids 1–1207), receptor-binding domain (RBD; amino acids 1–15 and 318–542), N-terminal domain (NTD; amino acids 1–305) and S1 (amino acids 1–685). A stabilized soluble version of the spike protein was designed by inclusion of the proline substitutions at positions 986 and 987, and disruptive replacement of the furin cleavage site RRAR (residues at position 682-685) with GSAS, as reported^30,31. C-terminal his-tag as well as streptag, was included in all constructs in order to facilitate protein purification. Expression of the recombinant proteins was performed using ExpiCHO^TM Expression system (Thermoscientific, USA) following purification using HisTrap^TM (GE Healthcare, UK) and Strep-Tactin^®XT (IBA, Germany).

In addition, huFc-RBD fused protein was expressed using previously designed Fc-fused protein expression vector³², giving rise to a protein comprising of two RBD moieties (amino acids 318–542, see accession number of the S protein above) owing to the homodimeric human (gamma1)Fc domain (huFc). Expression of the recombinant proteins performed using ExpiCHO^TM Expression system (Thermoscientific, USA) following purification using HiTrap Protein-A column (GE healthcare, UK). All purified proteins preserved in PBS.