title: Formation of functional peptide complexes of class II major histocompatibility complex proteins from subunits produced in Escherichia coli.
abstract: Class II major histocompatibility complex molecules play a major role in the immune response by binding peptide fragments of exogenous antigens and displaying them on the surfaces of antigen-presenting cells, where they can be recognized by T cells. To facilitate structural and functional studies of these molecules, we have produced truncated alpha and beta chains of the murine class II molecule I-Ek in Escherichia coli (Ec-I-Ek) and have developed conditions to fold them in the presence of specific peptides with yields of complex approaching 2%. Reconstitution is specific since only unlabeled peptide known to bind I-Ek compete with biotinylated peptide, as assessed by ELISA. Complexes of the refolded heterodimer (Ec-I-Ek) with either of two different peptide antigens remain associated during nonreducing SDS/PAGE. Immobilized Ec-I-Ek-peptide complexes stimulate lymphokine production by three T-cell clones in an antigen-specific manner with a dose-response relation comparable to previously described soluble I-Ek molecules produced in CHO cells. These results demonstrate that folding of Ek alpha and Ek beta polypeptides does not require any other protein to produce the biologically relevant conformation and that carbohydrate modification of this class II molecule is not necessary for alpha beta T-cell recognition.
authors.affiliation: Department of Microbiology and Immunology, Stanford University, CA 94305-5428.
authors.forename: J D
authors.initials: JD
authors.lastname: Altman
authors.forename: P A
authors.initials: PA
authors.lastname: Reay
authors.forename: M M
authors.initials: MM
authors.lastname: Davis
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D003001:Cloning, Molecular; D003574:Cytochrome c Group / Q000276:immunology / Q000378:metabolism; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D000949:Histocompatibility Antigens Class II / Q000096:biosynthesis / Q000378:metabolism*; D007700:Kinetics; D008213:Lymphocyte Activation; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology
chemical_list: D003574:Cytochrome c Group; D017931:DNA Primers; D000949:Histocompatibility Antigens Class II; D046911:Macromolecular Substances; D010455:Peptides; D011994:Recombinant Proteins