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-2Kb/TRP2180–188) was immobilized onto an ion-exchange chromatography column, and the β2m or TRP2180–188-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 24 h with a refolding yield of over 20%. Anti-H-2Kb mAb staining and flow cytometric analyses revealed that the on-column refolded H-2Kb/TRP2180–188 complexes had conformational characteristics similar to the dilution refolded H-2Kb/TRP2180–188 complexes and the commercial ones. Furthermore, H-2Kb/TRP2180–188 tetramer staining and the enumeration of TRP2180–188-specific T cells and H-2Kb-alloreactive T cells confirmed that the H-2Kb/TRP2180–188 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.

Research highlights
► A novel one-step chromatographic refolding method was developed.
► H-2Kb/TRP2180–188 complexes were prepared within 24 h with a high yield.
► On-column refolded complexes had structural conformations similar to traditional ones.
► On-column refolded complexes had TCR-binding ability similar to traditional ones.