Biotin ligase

Recombinant DRB4 protein was produced essentially as previously described (8 (link)). Briefly, DRB4 was purified from insect cell culture supernatants by affinity chromatography and dialyzed against phosphate storage buffer, pH 6.0. The protein was biotinylated at a sequence-specific site using biotin ligase (Avidity, Denver, CO) and dialyzed into phosphate storage buffer. The biotinylated monomer was loaded with 0.2 mg/ml of peptide by incubating at 37°C for 72 hours in the presence of 2.5 mg/ml n-octyl-β-D-glucopyranoside and 1 mM Pefabloc SC protease inhibitor mix (Sigma-Aldrich, St. Louis, MO). Peptide loaded monomers were subsequently conjugated as tetramers using R-PE streptavidin (Biosource International, Camarillo, CA) at a molar ratio of 8 to1.

Dextramer reagents comprised of IA^s/PLP 139-151 and IA^s/TMEV 70-86 (control) were generated as described previously [12 (link)]. We have used IA^s/TMEV 70-86 dextramers as controls to ascertain TCR-binding specificity of IA^s/PLP 139-151 dextramers, in all dextramer staining reactions [12 (link)]. Briefly, the α and β constructs of IA^s allele along with the peptide of interest was expressed together using baculovirus expression systems in SF9 insect cells (Invitrogen, Carlsbad, CA). Soluble MHC class II monomers of IA^s were then purified, concentrated, and biotinylated using biotin ligase (25 μg/10 nmol of substrate; Avidity, Denver, CO) [12 (link), 14 (link), 15 (link)]. The biotinylated monomers were assembled to fluorophore conjugated dextran molecules (kindly provided by Immudex, Copenhagen, Denmark) at a molar ratio of 20:1 in 1x Tris HCl 0.05 M, pH 7.2, by incubating in the dark for 30 minutes at room temperature (RT) [12 (link)]. The dextramer reagents were aliquoted and stored at 4°C until use.

Recombinant DR0401 protein was produced as previously described (19 (link)). Soluble DR0401 was purified from insect cell culture supernatants and biotinylated at a sequence-specific site using biotin ligase (Avidity) prior to dialysis into phosphate storage buffer. The biotinylated monomer was loaded with 0.2 mg/ml of peptide by incubating at 37°C for 72 hours in the presence of 2.5 mg/ml n-octyl-β-D-glucopyranoside and 1 mM Pefabloc SC (Sigma-Aldrich). Peptide loaded monomers were conjugated into tetramers using R-PE streptavidin (Invitrogen) at a molar ratio of 8 to 1.