Bestbac 2.0 linearized baculovirus dna

HLA-DM and HLA-DR401 were expressed recombinantly in High Five insect cells (species Trichoplusia ni; Thermo Fisher B85502) using a baculovirus expression system, as previously described (Birnbaum et al., 2014 (link); Rappazzo et al., 2020 (link)). Ectodomain sequences of each chain were formatted with a C-terminal poly-histidine purification tag and cloned into pAcGP67a vectors. Each vector was individually transfected into Sf9 insect cells (species Spodoptera frugiperda; Thermo Fisher 11496015) with BestBac 2.0 linearized baculovirus DNA (Expression Systems; Davis, CA) and Cellfectin II Reagent (Thermo Fisher), and propagated to high titer. Viruses were co-titrated for optimal expression to maximize balanced MHC heterodimer formation, co-transduced into High Five cells, and grown for 48–72 hr at 27°C. The secreted protein was purified from pre-conditioned media supernatant with Ni-NTA resin and purified via size exclusion chromatography with an S200 increase column on an AKTA PURE FPLC (GE Healthcare). To improve protein yields, the HLA-DRB1*04:01 chain was expressed with a CLIP_87-101 peptide (PVSKMRMATPLLMQA) connected to the N-terminus of the MHC chain via a flexible, 3C protease-cleavable linker.

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.