Conformational integrity and ligand binding properties of a single chain T-cell receptor expressed in Escherichia coli.
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.
Khandekar
S S
SS
Procept, Inc., Cambridge, Massachusetts 02139, USA.
Bettencourt
B M
BM
Wyss
D F
DF
Naylor
J W
JW
Brauer
P P
PP
Huestis
K
K
Dwyer
D S
DS
Profy
A T
AT
Osburne
M S
MS
Banerji
J
J
Jones
B
B
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*
D000949:Histocompatibility Antigens Class II; D008024:Ligands; D010455:Peptides; D011948:Receptors, Antigen, T-Cell; D011994:Recombinant Proteins; D018089:Superantigens