Bira biotin protein ligase reaction kit

SARS CoV-2 Spike trimer (S-2P) and subdomains (NTD, RBD-SD1, S1) were produced by transient transfection of 293 Freestyle cells as previously described (4 (link)). Avi-tagged S1 was biotinylated using the BirA biotin-protein ligase reaction kit (Avidity, #BirA500) according to the manufacturer’s instructions. The S-2P, RBD-SD1, and NTD proteins were produced by an in-column biotinylation method as previously described (5 (link)). Successful biotinylation was confirmed using Bio-Layer Interferometry, by testing the ability of biotinylated protein to bind to streptavidin sensors. Retention of antigenicity was confirmed by testing biotinylated proteins against a panel of cross-reactive SARS-CoV and SARS CoV-2 human monoclonal antibodies. Biotinylated probes were conjugated using either allophycocyanin (APC)-, Ax647-, BV421-, BV786-, BV711-, or BV570-labeled streptavidin. Reactions were prepared at a 4:1 molecular ratio of biotinylated protein to streptavidin, with every monomer labeled. Labeled streptavidin was added in ⅕ increments and in the dark at 4°C (rotating) for 20 min in between each addition. Optimal titers were determined using splenocytes from immunized mice and validated with SARS CoV-2 convalescent human PBMC.

To generate biotinylated S-protein probes for B cell sorting, coronavirus S-proteins were constructed with an Avi-tag at the C terminus. The Avi-tagged S-proteins were concentrated to 7 to 9 mg/ml in tris-buffered saline (TBS) before the biotinylation reaction using the BirA Biotin-Protein Ligase Reaction Kit (Avidity) following the manufacturer’s instructions. Briefly, for each reaction, 50 μl of protein solution, 7.5 μl of BioB Mix, 7.5 μl of Biotin200, and 5 μl of BirA ligase (3 mg/ml) were added in PCR tubes and mixed thoroughly. After incubating on ice for 3 hours, the biotinylated protein was purified by size exclusion chromatography. The biotinylated proteins were evaluated by BLI using the SA biosensor.