Hispure ni nta resin

The genes coding for FliD_cj, FliD_sm, and FliD_pa, codon-optimized for expression in E. coli, were synthesized (BioBasic) and sub-cloned into pET28a (Novagen) (Supplementary Table 4). Recombinant proteins were expressed in E. coli BL21-CodonPlus(DE3)-RIL cells containing the corresponding plasmids. For FliD_cj, transformants were grown in LB medium at 37 °C until they reached log phase, and expression was induced by the addition of 1 mM IPTG overnight at 20 °C. For both FliD_pa and FliD_sm, expression was auto-induced in ZYM-5052^{33 (link)} media at 20 °C overnight. For all three proteins, cells were collected by centrifugation, resuspended in 50 mM HEPES 150 mM NaCl pH 7 and sonicated. The lysate was centrifuged at 14,000g at 4 °C for 45 min. The supernatants were applied onto a 5 ml HisPure™ Ni-NTA resin (ThermoScientific) gravity-based column equilibrated with 50 mM HEPES 150 mM NaCl pH 7 and eluted using a linear 20–500 mM Imidazole gradient. Fractions containing FliD were pooled and applied to a HiLoad Superdex 200 16/600 column (GE Healthcare) equilibrated with 50 mM HEPES 150 mM NaCl pH 7 for FliD_cj and 50 mM Tris 150 mM NaCl pH 8 for FliD_pa and FliD_sm.

The RBD antigen was prepared as described [8 ]. Briefly, The RBD sequence is based on the genomic sequence of the first virus isolated Wuhan-Hu-1 and optimized for mammalian cell expression. The natural Spike N terminal signal peptide (amino acids 1–14) was fused to the RBD amino acids 319–541 and also fused with a C-terminal hexahistidine tag to allow isolation. The recombinant protein was produced in Expi293F cells (Thermo fisher) by transfecting these cells with purified DNA using Expifectamine 293 kit (Thermo fisher). Three days post-transfection the cells were harvested and the RBD protein was isolated from the supernatant by 2 hours incubation at RT with HisPure Ni-NTA resin (Thermo). The RBD was eluted and dialyzed against PBS.