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.
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.
Denkberg
G
G
Faculty of Biology, Technion-Israel Institute of Technology, Haifa, Israel.
Cohen
C J
CJ
Segal
D
D
Kirkin
A F
AF
Reiter
Y
Y
D000951:Antigens, Neoplasm / Q000737:chemistry / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics; D016356:Gene Products, tax / Q000737:chemistry / Q000276:immunology; D015789:HLA-A2 Antigen / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D013058:Mass Spectrometry; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
D000951:Antigens, Neoplasm; D016356:Gene Products, tax; D015789:HLA-A2 Antigen; D011994:Recombinant Proteins