Production and Purification of Stabilized SARS-CoV-2 Spike Proteins

Soluble 6P-stabilized SARS-CoV-2 spike proteins (WT, BA.1, BA.2, BA.3 and BA.4/5) were expressed by transient transfection^{20 (link),36 (link)}. In brief, the genes encoding spike ECD of different variants were synthesized and codon-optimized by GenScript, and then cloned into the mammalian expression vector pcDNA3.1 (Invitrogen). The plasmid was transfected using PEI into FreeStyle 293-F cells (Invitrogen). Transfected cells were cultured at 35 °C, 8% CO₂, and the cell culture supernatant was collected following 4 to 5 days of incubations. Protein was purified from filtered cell supernatants using Ni Sepharose resin (Cytiva) and further purified by gel filtration chromatography using a Superose 6 10/300 column (Cytiva) in 1 × TBS (20 mM Tris-HCl, 200 mM NaCl, pH8.0).