Recombinant Peptide-HLA-B*15:01 Complex Production

pET-30a(+) DNA plasmids encoding HLA-B*15:01 α-chain and human β2-microglobulin were transformed separately into BL21 E. coli and expressed. Inclusion bodies containing the individually expressed recombinant proteins were extracted, purified, and quantified as previously described.^{35 (link)} Soluble peptide-HLA-B*15:01 complexes were produced by refolding 30 mg of HLA-B*15:01 α-chain with 10 mg of β2-microglobulin and 5 mg of peptide (Genscript, Piscataway, USA) into a buffer of 0.5 M L-Arginine (Sigma-Aldrich, St Louis, USA), 0.1 M Tris-HCl pH 8.0 (Fisher Bioreagents, Waltham, USA), 2.5 mM EDTA pH 8.0 (Sigma-Aldrich, St Louis, USA), 5 mM Glutathione (reduced) (Goldbio, St Louis, USA), 1.25 mM Glutathione (Goldbio, St Louis, USA). The peptides selected are summarized in Table 1. The refold mixture was dialyzed at 4°C in 10 mM Tris-HCl pH 8.0 (3 times for 12 hours) and soluble pHLA complexes were purified via anion exchange chromatography.