title: Conformational integrity and ligand binding properties of a single chain T-cell receptor expressed in Escherichia coli.
abstract: We recently showed that a soluble, heterodimeric murine D10 T-cell receptor (TCR) (Valpha2Calpha, Vbeta8.2Cbeta) expressed in insect cells binds both Vbeta8.2-specific bacterial superantigen staphylococcal enterotoxin C2 (SEC2) and a soluble, heterodimeric major histocompatibility complex class II I-Ak.conalbumin peptide complex with a low micromolar affinity. To define further the structural requirements for the TCR/ligand interactions, we have produced in Escherichia coli a soluble, functional D10 single chain (sc) TCR molecule in which the Valpha and Vbeta domains are connected by a flexible peptide linker. Purified and refolded D10 scTCR bound to SEC2 and murine major histocompatibility complex class II I-Ak.conalbumin peptide complex with thermodynamic and kinetic binding constants similar to those measured for the baculovirus-derived heterodimeric D10 TCR suggesting that neither the TCR constant domains nor potential N- or O-linked carbohydrate moieties are necessary for ligand recognition and for expression and proper folding of the D10 scTCR. Purified D10 scTCR remained soluble at concentrations up to 1 mM. Circular dichroism and NMR spectroscopy indicated that D10 scTCR is stabilized predominantly by beta-sheet secondary structure, consistent with its native-like conformation. Because of its limited size, high solubility, and structural integrity, purified D10 scTCR appears to be suitable for structural studies by multidimensional NMR spectroscopy.
authors.affiliation: Procept, Inc., Cambridge, Massachusetts 02139, USA.
authors.forename: S S
authors.initials: SS
authors.lastname: Khandekar
authors.forename: B M
authors.initials: BM
authors.lastname: Bettencourt
authors.forename: D F
authors.initials: DF
authors.lastname: Wyss
authors.forename: J W
authors.initials: JW
authors.lastname: Naylor
authors.forename: P P
authors.initials: PP
authors.lastname: Brauer
authors.forename: K
authors.initials: K
authors.lastname: Huestis
authors.forename: D S
authors.initials: DS
authors.lastname: Dwyer
authors.forename: A T
authors.initials: AT
authors.lastname: Profy
authors.forename: M S
authors.initials: MS
authors.lastname: Osburne
authors.forename: J
authors.initials: J
authors.lastname: Banerji
authors.forename: B
authors.initials: B
authors.lastname: Jones
mesh_terms: D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D000949:Histocompatibility Antigens Class II / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D011487:Protein Conformation; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018089:Superantigens / Q000378:metabolism*
chemical_list: D000949:Histocompatibility Antigens Class II; D008024:Ligands; D010455:Peptides; D011948:Receptors, Antigen, T-Cell; D011994:Recombinant Proteins; D018089:Superantigens