D biotin

To detect antigen-specific cells, AP205-AF647 was generated by conjugating AF647 to the purified AP205 VLPs using the AF647 labeling kit from Thermo Fisher Scientific as described before.³³ (link) Qb-AF647³³ (link) was used to indicate potential AF647⁺ cells.
The DNA sequence encoding the BirA (Gene ID: 948469) was synthesized and cloned into the pGEX vector (Addgene). E. coli BL21 (DE3) transformed with pGEX-BirA was grown in LB medium and protein expression was induced with 0.1 mM IPTG at 18°C for 18 h. GST-BirA protein was purified by affinity chromatography using GST Fusion Protein Purification Kit (GenScript,Cat.NO L00206) according to the manufacturer’s instructions. The biotinylated RBD was generated by mixing 100 μM RBD-AviTag, 1 μM GST-BirA, 10mM magnesium chloride, 10mM ATP, and 150 μM D-Biotin (BBI Life Sciences, China). The mixture was incubated at 30°C for 1 h as described before.⁷⁹ (link) Biotinylated RBD was then mixed with fluorochrome-conjugated streptavidin at room temperature for 60 min to generate fluorescently labeled RBD tetramer.