Bira biotin protein ligase

Disulfide-stabilized HLA complexes were generated as previously described²⁴ . In short, HLA-A*02:01 heavy chain with disulfide-introducing mutations C-terminal BirA signal sequences or His6-Tag and human $\beta$ 2m light chain was produced in E. coli as inclusion bodies and purified. Heavy chain, $\beta$ 2m light chain, and GM dipeptide were diluted in refolding buffer [100 mM tris-Cl (pH 8), 0.5 M arginine, 2 mM EDTA, 0.5 mM oxidized glutathione, and 5 mM reduced glutathione] and incubated for 2 to 8 days at 4  ${}^{\circ }$ C while stirring before concentration. The concentrated protein was purified by SEC in 20 mM tris-HCl (pH 8)/150 mM NaCl on an ÄKTAprime system (GE Healthcare) using a HiLoad 26/600 Superdex 200 pg column (GE Healthcare). Peak fraction was either concentrated directly to 2000 $\mathrm{\mu }$ g/ml, aliquoted and frozen at -80 ${}^{\circ }$ C or biotinylated by BirA biotin protein ligase (Avidity) overnight at 4  ${}^{\circ }$ C according to the manufacturer’s instructions, and subjected to a second gel filtration before final concentration to 2000 $\mathrm{\mu }$ g/ml, aliquoting and storage at -80  ${}^{\circ }$ C.

Synthetic nanobodies were selected against OX₂R following procedures described previously⁵⁷ . Engineered OX₂R, carrying an additional amino-terminal Avi-tag, was expressed and purified in the presence of 50 µM small-molecule agonist as described above and biotinylated using BirA biotin-protein ligase (Avidity LLC) according to the manufacturer’s instructions. Binders to the immobilized receptor were selected from the three synthetic nanobody libraries, constructed by Zimmermann and coworkers⁵⁷ , by ribosome display followed by phage display as described⁵⁷ . Two rounds of selection were performed in the presence of 50 µM small-molecule agonist. In a third round, selections were competed with an excess of unbiotinylated OX₂R in the absence of agonist to enrich high-affinity binders against agonist-bound OX₂R. A total of 570 clones were tested by ELISA as described⁵⁷ , using an unrelated membrane protein to eliminate nonspecific binders. Sb51 was identified from a set of 57 unique hits based on expression level, sample homogeneity after purification, and affinity for agonist-bound OX₂R (Supplementary Data 1). The dissociation constant K_D for the binding of Sb51 to agonist-bound OX₂R was ~0.6 nM as determined by grating-coupled interferometry on a WAVE system (Creoptix), using OX₂R immobilized via the biotinylated Avi-tag on a neutravidin-coated WAVEchip 4PCP (Creoptix).