(A) Peptide-deficient HLA-B can be prepared by cleavage of engineered HLA-B (LZ-ELBM) with a specific enzyme and peptide-loaded versions by subsequent incubation with specific peptide as described in the scheme. (B) Representative SDS (left panels) and native (right panels)-PAGE gels showed cleavage and loading of B*18:01, B*35:01, B*44:02, and B*51:01 molecules with peptides DEVASTHDW (DW9), HPVGEADYFEY (HY11), EEIPDFAFY (EY9) and VPYEPPEV (VV8), respectively. (C) Averaged (n ≥ 3 replicates) normalized thermal shift assays were performed with peptide-deficient conformers of B*18:01, B*35:01, B*44:02 and B*51:01 molecules. B*18:01 and B*35:01 are more stable than B*44:02 and B*51:01.