title: Characterization of MHC/peptide complexes refolded by a one-step ion-exchange chromatography.
abstract: The current refolding process of MHC/peptide complexes is low-yield and time-consuming, thereby limiting the wide uses of MHC/peptide multimers. Here the heavy chain protein of MHC/peptide complex (H-2K(b)/TRP2(180-188)) was immobilized onto an ion-exchange chromatography column, and the β2m or TRP2(180-188)-fused β2m protein, which renatured previously in refolding buffer, was able to pass through the column for the gradient refolding. This strategy refolds, concentrates and purifies MHC/peptide complexes in a single integrated step, achieving a high level of process simplification and automation. Using this on-column refolding method, MHC/peptide complexes could be prepared within 24h with a refolding yield of over 20%. Anti-H-2K(b) mAb staining and flow cytometric analyses revealed that the on-column refolded H-2K(b)/TRP2(180-188) complexes had conformational characteristics similar to the dilution refolded H-2K(b)/TRP2(180-188) complexes and the commercial ones. Furthermore, H-2K(b)/TRP2(180-188) tetramer staining and the enumeration of TRP2(180-188)-specific T cells and H-2K(b)-alloreactive T cells confirmed that the H-2K(b)/TRP2(180-188) complexes prepared by on-column refolding or dilution refolding had comparable TCR-binding ability. These data demonstrate a novel, simple and efficient refolding strategy for the generation of MHC class I/peptide complexes.
authors.affiliation: Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Southeast University School of Medicine, Nanjing, Jiangsu, China.
authors.forename: Chuanlai
authors.initials: C
authors.lastname: Shen
authors.forename: Yong
authors.initials: Y
authors.lastname: He
authors.forename: Fanyan
authors.initials: F
authors.lastname: Meng
authors.forename: Kai
authors.initials: K
authors.lastname: Cheng
authors.forename: Daoping
authors.initials: D
authors.lastname: Zhang
authors.forename: Fengqin
authors.initials: F
authors.lastname: Miao
authors.forename: Jianqiong
authors.initials: J
authors.lastname: Zhang
mesh_terms: D000818:Animals; D002852:Chromatography, Ion Exchange / Q000379:methods*; D006649:Histocompatibility Antigens / Q000032:analysis* / Q000276:immunology; D008297:Male; D051379:Mice; D010455:Peptides / Q000276:immunology*; D058849:Protein Refolding; D011948:Receptors, Antigen, T-Cell / Q000276:immunology
chemical_list: D006649:Histocompatibility Antigens; D010455:Peptides; D011948:Receptors, Antigen, T-Cell