Diverse approaches to isolate HLA class I molecules from bacterial inclusion bodies, forming heterotrimeric complexes

Production of recombinant human leukocyte antigen class I (HLA-I) proteins in vitro is fundamental for molecular immunology. However, HLA-I protein refolding has remained inefficient due to challenges in the assembling of the trimolecular complex. Here, we compare various in vitro refolding methods that address the challenges of intrachain disulfide bond formation and assembly of the complex between the light and heavy chains in the presence of the target peptide. We developed methods that uncouple the oxidation of disulfide bond formation of both subunits of HLA-I, followed by renaturation to promote complex formation. CuSO₄-catalyzed air oxidation enhances correct disulfide bond formation when the protein is solubilized with N-lauryl-sarcosine (sarkosyl); however, careful removal of sarkosyl did not prevent heavy chain aggregation. We modified the classical method of HLA-I refolding by pre-oxidizing the β₂m light chain before adding the HLA-I heavy chain and peptide. This method yielded successful complex refolding for HLA-A∗02:01/GILGFVFTL at 24.2 % efficiency, and HLA-C∗12:03/KAYNVTQAF at 14.5 % efficiency. Our results suggest that pre-folded β₂m improves refolding efficiency of HLA-I molecules. This work presents novel approaches to HLA-I refolding that may be applied to other difficult-to-fold protein complexes.