Recombinant MHC-II Protein Production

Recombinant soluble HLA-DM, HLA-DR401, and HLA-DR402 were produced in High Five (Hi5) insect cells (Thermo Fisher) via a baculovirus expression system, as previously described for other MHC-II proteins^{32 (link)}. Ectodomain sequences of each chain followed by a poly-histidine purification site were cloned into pAcGP67a vectors. For each construct, 2 µg of plasmid DNA was transfected into SF9 insect cells with BestBac 2.0 linearized baculovirus DNA (Expression Systems, Davis CA) using Cellfectin II reagent (Thermo Fisher, Waltham MA). Viruses were propagated to high titer, co-titrated to maximize expression and ensure 1:1 MHC heterodimer formation, then co-transduced into Hi5 cells and grown at 27 °C for 48–72 h. Proteins were purified from the pre-conditioned media supernatant with Ni-NTA resin and size purified via size exclusion chromatography using a S200 increase column on an AKTAPURE FPLC (GE Healthcare, Chicago IL). HLA-DRB1*04:01 and HLA-DRB1*04:02 chains were expressed with CLIP_81-101 peptide connected by a 3C-protease-cleavable flexible linker to the MHC N-terminus to improve protein yields.