Characterization of MHC/peptide complexes refolded by a one-step ion-exchange chromatography.

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.