Imidazole

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.

The pET-GST-P1(O97) plasmid was transferred to Escherichia coli (BL21) to express the recombinant GST-P1. The recombinant GST-P1 was purified from the total lysate of transformed Escherichia coli (BL21) by nickel-charged affinity resin (Ni Sepharose High Performance, Cytiva, Marlborough, MA, USA). The impurities were removed using phosphate-based buffer with 30 mM imidazole (Sigma-Aldrich, St. Louis, MO, USA). The recombinant GST-P1 was eluted using phosphate-based buffer with 250 mM imidazole. The imidazole was removed with a PD-10 desalting column (Cytiva, Marlborough, MA, USA).

Ran(Q69L), cloned into a pQE32 vector, was transformed into Escherichia coli competent cells (strain BL21-CodonPlus(DE3)-RIL). Expression was induced with 0.2 mM IPTG (Roth) at an optical density of 0.5–0.8 in 2xYT medium. Cells were grown at 30 °C for 16 h. Afterwards, cells were collected, washed with PBS and cell pellet was stored at −80 °C. The pellet was resuspended in lysis buffer (10 mM HEPES pH 7.6, 100 mM KCl, 1 mM MgCl₂, 5% v/v glycerol, 1 mM DTT, 0.1% Triton X-100, cOmplete EDTA-free protease inhibitor cocktail (Roche) and PMSF), lysed by sonification and clarified by centrifugation at 40,000 rpm for 20 min in a Type 50.2 Ti Rotor (Beckman Coulter). Supernatant was incubated with Ni-NTA Agarose beads (Qiagen) for 2 h at 4 °C, washed with lysis buffer and eluted with lysis buffer supplemented with 300 mM imidazole (Roth). imidazole was removed via buffer exchange through a HiTrap Desalting column (5 mL, Cytiva) on an ÄKTA go system (Cytiva) operated using the UNICORN software (version 7.6). Ran(Q69L) was concentrated to 350 μM and stored at −80 °C.