Spodoptera frugiperda

MHCII monomers were produced in High Five insect cells (Trichoplusia ni BTI-Tn-5B1-4 cells, Invitrogen) using the baculovirus expression system^{40 (link),54 (link),55 (link)}. DNA encoding the I-A^b α- and β-chains and the mouse MPO_415–428 (₄₁₅KLYQEARKIVGAMV₄₂₈), fused to the N-terminus of the β-chain via a flexible linker (SGGSGSGSAS), were cloned into pFastBac Dual vector and recombinant baculovirus propagated in Sf9 insect cells (Spodoptera frugiperda, Invitrogen). The C-termini of the I-A^b α- and β-chains contained enterokinase cleavable Fos and Jun leucine zippers, respectively, to promote correct heterodimeric pairing. The C-terminus of the β-chain also contained a BirA ligase recognition sequence for biotinylation and poly-histidine tag for purification, immediately following the Jun leucine zipper sequence. MPO:I-A^b monomers were purified from baculovirus infected High Five insect cell supernatants through immobilized metal ion affinity (Ni Sepharose 6 Fast-Flow, GE Healthcare), size exclusion (S200 Superdex 16/600, GE Healthcare) and anion exchange (HiTrap Q, HP, GE Healthcare) chromatography. MPO:I-A^b tetramers were assembled by the addition of Streptavidin-PE (BD Biosciences)^{54 (link),55 (link)}.

Codon-optimized synthetic genes for expression in Spodoptera frugiperda (Invitrogen GeneArt), coding for Arabidopsis thaliana MIK2 (residues 1–709) and BAK1 (1–220) ectodomains were cloned into a modified pFastBac (Geneva Biotech) vector, providing a TEV (tobacco etch virus protease) cleavable C-terminal StrepII-9xHis tag. For protein expression, Trichoplusia ni Tnao38 cells^{49 (link)} were infected with MIK2 or BAK1 virus with a multiplicity of infection (MOI) of 3 and incubated for 1 d at 28 °C and 2 d at 22 °C at 110 rpm. The secreted proteins were purified from the supernatant by sequential Ni²⁺ (HisTrap Excel column; GE Healthcare; equilibrated in 25 mM KP_i pH 7.8, 500 mM NaCl) and StrepII (Strep-Tactin Superflow high capacity (IBA Lifesciences) equilibrated in 25 mM Tris pH 8.0, 250 mM NaCl, 1 mM EDTA) affinity chromatography. All proteins were incubated with TEV protease to remove the tags. Proteins were further purified by SEC on a Superdex 200 increase 10/300 GL column (GE Healthcare), equilibrated in 20 mM sodium citrate pH 5.0, 150 mM NaCl. For biochemical experiments, proteins were concentrated using Amicon Ultra concentrators (Millipore, molecular weight cutoff 10,000 and 30,000).

Sf9 cell line derived from the fall armyworm Spodoptera frugiperda was obtained from Invitrogen and maintained in Grace’s insect cell medium supplemented with 10% FCS, antibiotics (100 IU/mL penicillin, 100 μg/mL streptomycin) and 2.5 μg/mL amphotericin B at 27°C. The human promyelocytic leukemia cell line HL60 and murine plasmacytoma NS0 were obtained from American Type Culture Collection and maintained in RPMI 1640 medium containing 10% FCS, 100 IU/mL penicillin and 100 μg/mL streptomycin at 37°C and 5% CO₂.