title: Construction of bioactive chimeric MHC class I tetramer by expression and purification of human-murine chimeric MHC heavy chain and beta(2)m as a fusion protein in Escherichia coli.
abstract: Major histocompatibility (MHC) class I tetramers are used in the quantitative analysis of epitope peptide-specific CD8+ T-cells. An MHC class I tetramer was composed of 4 MHC class I complexes and a fluorescently labeled streptavidin (SA) molecule. Each MHC class I complex consists of an MHC heavy chain, a beta(2)-microglobulin (beta(2)m) molecule and a synthetic epitope peptide. In most previous studies, an MHC class I complex was formed in the refolding buffer with an expressed MHC heavy chain molecule and beta(2)m, respectively. This procedure inevitably resulted in the disadvantages of forming unwanted multimers and self-refolding products, and the purification of each kind of monomer was time-consuming. In the present study, the genes of a human/murine chimeric MHC heavy chain (HLA-A2 alpha1, HLA-A2 alpha2 and MHC-H2D alpha3) and beta(2)m were tandem-cloned into plasmid pET17b and expressed as a fusion protein. The recombinant fusion protein was refolded with each of the three HLA-A2 restricted peptides (HBc18-27 FLPSDFFPSI, HBx52-60 HLSLRGLPV, and HBx92-100 VLHKRTLGL) and thus three chimeric MHC class I complexes were obtained. Biotinylation was performed, and its level of efficiency was observed via a band-shift assay in non-reducing polyacrylamide gel electrophoresis (PAGE). Such chimeric MHC class I tetramers showed a sensitive binding activity in monitoring HLA/A2 restrictive cytotoxic T lymphocytes (CTLs) in immunized HLA/A*0201 transgenic mice.
authors.affiliation: Department of Medical Genetics, The Second Military Medical University, 200433 Shanghai, China.
authors.forename: Ding
authors.initials: D
authors.lastname: Ren
authors.forename: Fang
authors.initials: F
authors.lastname: Wang
authors.forename: Xiaowen
authors.initials: X
authors.lastname: He
authors.forename: Lei
authors.initials: L
authors.lastname: Jiang
authors.forename: Dean
authors.initials: D
authors.lastname: Li
authors.forename: He
authors.initials: H
authors.lastname: Ying
authors.forename: Shuhan
authors.initials: S
authors.lastname: Sun
mesh_terms: D000818:Animals; D019921:Biotinylation; D016827:CD8 Antigens / Q000276:immunology; D003001:Cloning, Molecular; D004926:Escherichia coli; D005260:Female; D015234:HLA-A Antigens / Q000235:genetics; D015789:HLA-A2 Antigen / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D015395:Histocompatibility Antigens Class I / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008822:Mice, Transgenic; D010957:Plasmids; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D001613:beta 2-Microglobulin / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*
chemical_list: D016827:CD8 Antigens; D015234:HLA-A Antigens; C435939:HLA-A*02:01 antigen; D015789:HLA-A2 Antigen; D015395:Histocompatibility Antigens Class I; D011993:Recombinant Fusion Proteins; D001613:beta 2-Microglobulin