BirA biotin-protein ligase standard reaction kit

To detect gp120-specific B cells by flow cytometry, we synthesized fluorescently labeled 1086.C gp120 for cell staining. Avi-tagged 1086.C gp120 was biotinylated using BirA biotin-protein ligase standard reaction kit (Avidity, BirA500) following the manufacturer’s protocol. Tetramers were prepared based on the molar ratio (4:1) of the analyte protein and fluorochrome-conjugated streptavidin, respectively. Alexa Fluor 647 (ThermoFisher, S21374) or Brilliant Violet 421 (BioLegend, 405225) conjugated streptavidin was reacted with the biotinylated protein over 5 additions, incubating for 15 min between each addition. The final concentration of tetramer was calculated with respect to the analyte protein after which PBS was added to achieve concentration of 3 µM. The solution was aliquoted, snap frozen, and stored at −80°C.

MS2 and S proteins were biotinylated in vitro with a BirA biotin-protein ligase standard reaction kit (Avidity LLC). Following buffer exchange into 20 mM Tris, 20 mM NaCl, pH 8 buffer, the proteins were concentrated to 45 μM, then BirA and a mixture of biotin, ATP, and magnesium acetate (Biomix B) was added to the protein solution. The solution was allowed to mix overnight at 4 °C. More Biomix B was added, and the solution was mixed at 37 °C for 2 h, followed by the addition of more Biomix B and another overnight incubation at 4 °C. The protein was then purified on a Superdex 200 Increase 10/300 column (Cytiva) to remove BirA and excess biotin and quantified by a BCA assay.

The in vitro folding of murine MHC class I molecules was performed using photocleavable ligand as described above and previously^{3 (link)}. Conditional UV ligands were purchased and synthesized from Leiden University Medical Center peptide synthesis unit (LUMC) following previously described methods^{31 (link)} To setup the refolding reaction, heavy chains (1 µM) and β2m (2 µM) were diluted in a folding buffer composed of 0.1 M Tris pH 8.0, 500 mM L-Arginine-HCl, 2 mM EDTA, 0.5 mM oxidized glutathione and 5 mM reduced glutathione with 60 µM respective photocleavable peptide (H-2Db: ASNEN-J-ETM, H-2 Kb: FAPGNY-J-AL, H-2Dd: RGPGRA-J-VTI, H-2Kd: IYSTV-J-SSL, H-2Ld: YPNVNIH-J-F) (Table 1). After folding for 3–5 days at 4 °C, the folded protein was upconcentrated with a 10 kDa cut-off membrane filter (Vivaflow-200; Sartorius, Cat#VS0601) and biotinylated using BirA biotin-protein ligase standard reaction kit (Avidity, LLC- Aurora, Colorado). Finally, folded biotinylated monomer complexes were purified with size exclusion chromatography using HPLC (Waters Corporation, USA), and aliquots were stored at − 80 °C until further use.

An expression
vector pEF-Bos containing PD-L1 was transfected into HEK 293T Lenti-X
293T cells with Lipofectamine 3000 (Life Technologies, L3000). Cells
were cultured in Advanced DMEM (ThermoFisher, 124291015) with 1X Penicillin/Streptomycin
(Fisher Scientific, 15140122) and 2X Glutamax (Fisher Scientific,
35050061). Media were collected after 2 days of culture. Protease
cocktail inhibitors were added (Millipore Sigma, 11697498001), and
proteins were extracted and concentrated using 3 kDa Amicon centrifugal
units (Millipore Sigma, UFC800396) through 5 successive 25 min spindowns
at 4 °C and 7830 rpm. PD-L1 was then purified via its coupled
6xHis tag with a Ni-NTA beads (Qiagen, 30210) nickel column and biotinylated
using BirA biotin-protein ligase standard reaction kit (Avidity, BirA500).
Buffer exchange to PBS was performed using 10 kDa snakeskin dialysis
tubing for 24 h with 2 L of PBS (500 mL for 3 h, 500 mL for 5 h, and
1 L for 16 h). Total protein concentration was determined using Bio-Rad
Protein Assay Dye Reagent Concentrate (Bio-Rad, #5000006). Anti PD-L1
antibody was used in a Western blot to verify protein identity (eBioscience,
14-5983-82). Streptavidin Alexa Fluor 488 (Invitrogen, S11223) immunoblot
staining was used to verify biotinylation.

Affinity between human ACE2 to SARS-CoV-2 S1–614D, S1–614G, S-614D or S-614G were evaluated using Octet RED96 instrument at 30°C with a shaking speed at 1000 RPM (ForteBio). Streptavidin biosensors (SA) (ForteBio) were used. S1 proteins were pre-biotinylated using EZ-Link NHS-PEG4-Biotin (ThermoFisher Scientific). Following 20 minutes of pre-hydration of SA biosensors and 1 minute of sensor check, 100 nM S1–614D or S1–614G in 10X kinetic buffer (ForteBio) were loaded onto surface of SA biosensors for 5 minutes. After 2 minutes of baseline equilibration, 10 minutes of association was conducted at 2.5 to 75 nM of human ACE2, followed by 20 minutes of dissociation in the same buffer, which was used for baseline equilibration. S proteins with Avi-tag were pre-biotinylated using BirA biotin-protein ligase standard reaction kit (Avidity). 25 nM S-614D or 15 nM S-614G in 10X kinetic buffer (ForteBio) were loaded onto surface of SA biosensors for 5 minutes. After 2 minutes of baseline equilibration, 5 minutes of association was used for 2 to 32 nM hACE2, followed by 10 minutes of dissociation in 10X kinetic buffer. The data were corrected by subtracting reference sample, 1:1 binding model with global fit was used for determination of affinity constants.