Refolding and purification of pMHC-I proteins

Human β₂m (1-100), the luminal domain of HLA-A*02:01(1-276), HLA-A*03:01(1-274), HLA-B*27:05 (1-277), and HLA-B*27:09 (1-277) were expressed in BL21(DE3) Escherichia coli as inclusion body and purified as described below⁵⁰: Cell pellets were resuspended with P1 lysis buffer (50 mM Tris, 1% (w/v) sodium deoxycholate, 1% (w/v) Triton X-100, 100 mM NaCl, 0.1% (w/v) NaN3, 0.5 mM MgCl₂, 10 mM DTT, 20 µg/mL DNase I, pH 7.5) and sonicated for 30 min on ice water. The sonicated lysate was centrifuged at the speed of 12,000×g for 10 min and the supernatant was trashed. The white pellet is the inclusion bodies. Inclusion bodies were first washed twice with P2 buffer (50 mM Tris, 100 mM NaCl, 0.5% Triton X-100, 0.5 mM EDTA, 0.15 (w/v) NaN3, 10 mM DTT, pH 7.5) and one time with PBS buffer by sonicating for 10 min, followed by centrifuging at the speed of 12000 g for 10 min each time. In the last step, inclusion bodies were resuspended with P3 buffer (20 mM Tris, 0.5 mM EDTA, 4 M urea, 10 mM DTT, pH 8.0), dissolved overnight while rotating, centrifuged at the speed of 12000 g for 10 min to remove the unsolved particles. The supernatant is the desired denatured protein and was shock frozen and stored at −80 °C until use. Corresponding heavy chain mutants were generated by standard site directed mutagenesis using heavy chain WT as template, primers used were listed in Supplementary Table ⁴.

pMHC-I proteins were obtained by refolding heavy chain, β₂m with high-affinity peptides at 4 °C as described below. Briefly: heavy chain, β2m and the corresponding restricted high-affinity peptide were refolded together in refolding buffer (0.4 M arginine, 0.1 M Tris, GSH:GSSG = 5 mM:0.5 mM, 2 mM EDTA, 0.5 mM PMSF, pH 8.0 for A*03:01 and A*02:01, pH 7.5 for B*27:05/09). For 1 L refolding, 10 mg peptide (photocleavable peptide photoKV9 for A*02:01 first need be dissolved in DMSO) was first added into pre-cooled refolding buffer, followed by dropwise adding 30 mg β2m. After stirring for 2 h at 4 °C, 30 mg heavy chain was dropwise added. The refolding bottle was then kept at 4 °C while stirring (for photocleavable peptides, the refolding bottle need be kept in dark) until purification. The refolding time varies for different allotypes: 3 days for A*02:01, 5 days for A*03:01, 7 days for B*27:09 (heavy chain equipped with or without a His-tag) and B*27:05 without His-tag, 14 days for B*27:05 when heavy chain bears a His-tag. Peptides used for refolding in this study were chemical synthetized (GL Biochem (shanghai) ltd. or DG peptides, China). Peptides’ sequence are also summarized in Supplementary Table ¹: KILGFVFJ*V (photoKV9) for A*02:01, A*02:01Y116D, A*02:01H114D, A*03:01D116Y and A*03:01R114H/D116Y, KLIETYFJ*K (photoKK9) for A*03:01, A*03:01D116Y, A*03:01R114/HD116Y and A*02:01Y116D, RRKWRRWJ*L (photoRL9) for B*27:05/09, B*27:05D116Y and B*27:05H114R, IRAAPPPJ*F (photoIF9) for B*27:05/09, where J* = 3-amino-3-(2-nitrophenyl)-propionic acid⁵⁰, RRKWRRWHL (RL9) for B*27:05/09. Refolded pMHC-I complexes were purified by size-exclusion chromatography (SEC) with a Superdex 200 increase 10/300 GL column in PBS buffer. Pure fractions were pooled, shock frozen and stored at −80 °C until use. The T_m of pMHC-I molecules (WT) before and after UV exposure are summarized in Supplementary Table ².