title: Characterization of a single-chain T-cell receptor expressed in Escherichia coli.
abstract: Despite progress in defining the nature of major histocompatibility complex products that are recognized by the T-cell antigen receptor, the binding properties and structure of the receptor have not been solved. The primary problem has been the difficulty in obtaining sufficient quantities of active receptor. In this report we show that a single-chain T-cell receptor gene can be expressed in Escherichia coli. The protein consists of the variable (V) regions of the alpha and beta chains (V alpha and V beta) encoded by the cytotoxic T-lymphocyte clone 2C (a H-2b anti-H-2d alloreactive cell line) linked by a 25-amino acid flexible peptide. Solubilized extracts that contain the 27-kDa V alpha 3V beta 8 protein are positive in solid-phase immunoassays with the anti-V beta 8 antibody KJ16 and the anti-clonotypic antibody 1B2. Approximately 1% of the protein can be specifically purified on a 1B2-conjugated column. These results indicate that a fraction of the protein is able to fold into a native conformation and that single-chain proteins should be useful not only as immunogens for eliciting anti-T-cell receptor antibodies but in the study of T-cell receptor structure and function.
authors.affiliation: Department of Biochemistry, University of Illinois, Urbana 61801.
authors.forename: W F
authors.initials: WF
authors.lastname: Hoo
authors.forename: M J
authors.initials: MJ
authors.lastname: Lacy
authors.forename: L K
authors.initials: LK
authors.lastname: Denzin
authors.forename: E W
authors.initials: EW
authors.lastname: Voss
authors.forename: K D
authors.initials: KD
authors.lastname: Hardman
authors.forename: D M
authors.initials: DM
authors.lastname: Kranz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002460:Cell Line; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods; D003602:Cytotoxicity, Immunologic; D004926:Escherichia coli / Q000235:genetics*; D012515:Mast-Cell Sarcoma / Q000276:immunology; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods; D011487:Protein Conformation; D011948:Receptors, Antigen, T-Cell / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000302:isolation & purification; D015183:Restriction Mapping; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D009838:Oligodeoxyribonucleotides; D011948:Receptors, Antigen, T-Cell; D011994:Recombinant Proteins