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.

G Denkberg; C J Cohen; D Segal; A F Kirkin; Y Reiter

doi:10.1002/1521-4141(2000012)30:12<3522::aid-immu3522>3.0.co;2-d

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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.

G Denkberg·C J Cohen·D Segal·A F Kirkin·Y Reiter

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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 beta2-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-gamma, 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.

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Antigens, Neoplasm Escherichia coli Gene Products, tax HLA-A2 Antigen Humans Mass Spectrometry Protein Folding Recombinant Proteins T-Lymphocytes, Cytotoxic Journal Article Research Support, Non-U.S. Gov't

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G Denkberg, C J Cohen, D Segal, A F Kirkin, Y Reiter. 2000. 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.. https://doi.org/10.1002/1521-4141(2000012)30%3A12%3C3522%3A%3Aaid-immu3522%3E3.0.co%3B2-d

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