Biotinylation of SARS-CoV-2 Spike and RBD

To produce biotinylated spike and receptor binding domain (RBD) protein, HEK-293F cells were seeded at 1 × 10⁶ cells/mL in Freestyle™ 293 Expression Medium (Gibco). The next day, a transfection mix was prepared (for 200 mL of cells) of 72 μg of spike-Avi-His tag or RBD-Avi-His tag plasmid and 18 μg of BirA plasmid³⁶ (link)^,88 (link) into 11 mL of Opti-MEM^TM, alongside 2 mL of PEI-Max® and 3 mL of 10 mM biotin, and left to incubate at 37°C 5% CO₂ in a shaking incubator for 7 days before harvesting for purification. The supernatant was purified using an imidazole (Sigma-Aldrich) buffer at a final concentration of 20 mM during binding to the His GraviTrap™ (Cytiva) column and 500 mM imidazole for elution. The eluted protein was then concentrated with a 100KD Amicon® Ultra concentrator (Millipore) and washed with PBS before quantification using a NanoDrop^TM. Biotinylated protein was then further purified through size exclusion chromatography using an AKTA™ pure system with a Superdex® 200 Increase 10/300 GL column (Sigma-Aldrich) to select for fractions containing trimeric spike or RBD protein.