Bira biotin protein ligase bulk reaction kit

Random biotinylation of S-proteins was conducted using EZ-Link NHS-PEG Solid-Phase Biotinylation Kit (Thermo Scientific #21440). In all, 10 µl dimethyl sulfoxide (DMSO) was added per tube for making concentrated biotin stock, 1 µl of which were diluted into 170 µl water before use. Coronavirus spike proteins were concentrated to 7–9 mg/ml using 100 K Amicon tubes in PBS, then aliquoted into 30 µl in PCR tubes. In all, 3 µl of the diluted biotin were added into each aliquot of concentrated protein and incubated on ice for 3 h. After reaction, buffer exchange for the protein was performed using PBS to remove excess biotin. BirA biotinylation of S-proteins was conducted using BirA biotin-protein ligase bulk reaction kit (Avidity). Coronavirus S-proteins with Avi-tags were concentrated to 7–9 mg/ml using 100 K Amicon tubes in tris-buffered saline (TBS), then aliquoted into 50 µl in PCR tubes. In all, 7.5 µl of BioB Mix, 7.5 µl of Biotin200, and 5 µl of BirA ligase (3 mg/ml) were added per tube. The mixture was incubated on ice for 3 h, followed by size-exclusion chromatography to segregate the biotinylated protein and the excess biotin. The extend of biotinylation was evaluated by BLI streptavidin biosensors.

Rhesus FcγRs were produced as described previously (Chan et al., 2016 (link)) and human FcγRs reformatted with a C-terminal GGG-AVI-His tag (GGGLNDIFEAQKIEWHEHHHHHH) in order to allow site-specific enzymatic biotinylation. Expression and purification were carried out as described for the non-tagged variants. Biotinylation was carried out according to the conditions of the BirA biotin-protein ligase bulk reaction kit (Avidity). Briefly, 1 mg of FcγR after SEC purification (in PBS) was diluted to ~ 40 μM. A 1/8 volume of the diluted FcγR of each reagent Biomix A and Biomix B were added to the FcγR, followed by 1 vial (10 μL at 3 μg/μL) of BirA enzyme. The reaction was allowed to proceed for 2 h at 30 °C with end-over-end mixing. After biotinylation, the reaction mixture was buffer exchanged 3 × into PBS using 3 kD cutoff centrifugal filter units (Amicon UFC900396). Biotinylated FcγRs were detected at 280 nm to determine their concentration, and stored at − 80 °C for up to 6 months prior to use.

To produce g140 foldon-type trimers of SIVmac251 (GenBank# AJP75601.1) and SIVagm (agm.Sab92018, GenBank# ADO34206.1) Env glycoproteins, corresponding codon-optimized DNA fragments designed based on the original construct coding for uncleaved YU-2 trimers^{64 (link)} were synthesized (Genscript), and cloned into pcDNA™3.1/Zeo⁽⁺⁾ expression vector (Thermo Fisher Scientific). Trimeric SIV GP140 proteins were produced by transient transfection of FreeStyle™ 293-F cells using the PEI method^{65 (link)}, and purified by high-performance chromatography using the Ni Sepharose® Excel Resin according to manufacturer’s instructions (GE Healthcare). Proteins were controlled for purity by SDS-PAGE and NativePAGE gel staining as previously reported^{65 (link)}, and then biotinylated using BirA biotin-protein ligase bulk reaction kit (Avidity, LLC). Biotinylated SIV GP140 trimers were dialyzed against PBS using Slide-A-Lyzer® Cassettes (35 K MWCO, Thermo Fisher Scientific), and final protein concentrations were measured using a NanoDrop 2000 instrument (Thermo Fisher Scientific).