Plasmid Design and Cell Culture.
The SARS-CoV-2 D614G spike protein ectodomain expression plasmid was designed as follows: amino acids 1-1208, furin cut site PRRAR substituted to PGSAS, 6 HexaPro stabilization substitutions (K986P, V987P, F817P, A892P, A899P, A924P), a T4 fold self-trimerization domain, and a polyhistidine tag for purification by nickel-charged nitrilotriacetic acid (Ni-NTA). FASTA sequences for 614G and Omicron spike constructs are in Supporting Information. Expi293F cells were obtained from ThermoFisher as part of the Expi293F protein expression system. Cells were cultured for 3+ passages at 37°C/125 RPM/8% CO2 not exceeding a density of 5 × 106cells/ml or passage >30, seeded at a density of 2.5 × 106 cells/mL one day prior to transfection, and diluted to 3 × 106 cells/mL immediately before transfection. Plasmid was transfected at 1 μg DNA per 1 mL of cell culture using ExpiFectamine and Opti-Mem I Reduced Serum Medium (Thermo Scientific). Transfected culture was left to express for four days at 30°C/125 RPM/8% CO2 before harvesting and centrifuging twice at 4°C/3000 RCF for 10 minutes to clarify the supernatant.