Recombinant human single-chain MHC-peptide complexes made from E. coli by in vitro refolding: Functional single-chain MHC-peptide complexes and tetramers with tumor associated antigens

Galit Denkberg, Cyril J. Cohen, Dina Segal, Alexei F. Kirkin, Yoram Reiter

Research output: Contribution to journalArticlepeer-review

51 Scopus citations

Abstract

Soluble recombinant MHC-peptide complexes are valuable tools for molecular characterization of immune responses as well as for other functional and structural studies. In this study, soluble recombinant single-chain human MHC (scMHC)-peptide complexes were generated by in vitro refolding of inclusion bodies from bacterially expressed engineered HLA-A2 in the presence of tumor-associated or viral peptides. The scMHC molecule was composed of β2-microglobulin connected to the first three domains of the HLA-A2 heavy chain through a 15-amino acid flexible linker. Highly purified scMHC-peptide complexes were obtained in high yield using several peptides derived from the melanoma antigens gp100 and MART-1 or a viral peptide derived from HTLV-1. The scMHC complexes were characterized in detail and were found to be correctly folded and able to specifically bind HLA-A2-restricted peptides. We also generated scMHC-peptide tetramers, which were biologically functional; they induced a peptide-specific CTL clone to be activated and secrete IFN-γ, and were able to stain specifically CTL lines. Such recombinant soluble scMHC-peptide complexes and tetramers should prove of great value for characterization of immune responses involving CTL, for visualization of antigen-specific immune responses, for in vitro primary CTL induction, and for peptide binding assays and structural studies.

Original languageEnglish
Pages (from-to)3522-3532
Number of pages11
JournalEuropean Journal of Immunology
Volume30
Issue number12
DOIs
StatePublished - Dec 2000
Externally publishedYes

Keywords

  • Cancer peptide
  • MHC class I
  • MHC tetramer

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