Destabilizing single chain major histocompatibility complex class I protein for repurposed enterokinase proteolysis

Sci Rep. 2020 Sep 10;10(1):14897. doi: 10.1038/s41598-020-71785-2.


The lack of a high throughput assay for screening stabilizing peptides prior to building a library of peptide-major histocompatibility complex class I (pMHC-I) molecules has motivated the continual use of in silico tools without biophysical characterization. Here, based on de novo protein fragmentation, the EASY MHC-I (EZ MHC-I) assay favors peptide antigen screening to an unheralded hands-on time of seconds per peptide due to the empty single chain MHC-I protein instability. Unlike tedious traditional labeling- and antibody-based MHC-I assays, repurposed enterokinase directly fragments the unstable single MHC-I chain protein unless rescued by a stabilizing peptide under luminal condition. Herein, the principle behind EZ MHC-I assay not only characterizes the overlooked stability as a known better indicator of immunogenicity than classical affinity but also the novel use of enterokinase from the duodenum to target destabilized MHC-I protein not bearing the standard Asp-Asp-Asp-Asp-Lys motif, which may protend to other protein instability-based assays.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Enteropeptidase / metabolism*
  • Histocompatibility Antigens Class I / chemistry*
  • Histocompatibility Antigens Class I / metabolism*
  • Humans
  • Peptide Fragments / metabolism*
  • Protein Binding
  • Protein Stability
  • Proteolysis


  • Histocompatibility Antigens Class I
  • Peptide Fragments
  • Enteropeptidase