Peptide-MHC Class I Monomer Preparation

Peptide–MHC class I monomers refolded with an ultraviolet light (UV)-cleavable conditional ligand were prepared in-house as previously described (7 (link)). Briefly, recombinant H-2K^b heavy chain and human β2 microglobulin light chain were produced in Escherichia coli as inclusion bodies and refolded in the presence of a UV-cleavable peptide (SIINFE-J-L, where J represents 3-amino-3-(2-nitro) phenyl-propionic acid (Peptide 2.0). Monomers were captured by anion exchange (HiTrap Q HP, GE), biotinylated, and purified by gel filtration FPLC. UV-induced ligand exchange with mutant Lama4 (mLama4) peptide was performed as described previously (7 (link)). OVA-I (SIINFEKL)-H-2K^b-PE and mutant Lama4-H-2K^b-PE tetramers were additionally obtained from the Baylor College of Medicine MHC Tetramer Production Facility.