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Volume 272, Number 51, Issue of December 19, 1997 pp. 32190-32197

Conformational Integrity and Ligand Binding Properties of a Single Chain T-cell Receptor Expressed in Escherichia coli

(Received for publication, July 23, 1997, and in revised form, October 9, 1997)

From Procept, Inc., Cambridge, Massachusetts 02139

We recently showed that a soluble, heterodimeric murine D10 T-cell receptor (TCR) (V2C, V8.2C) expressed in insect cells binds both V8.2-specific bacterial superantigen staphylococcal enterotoxin C2 (SEC2) and a soluble, heterodimeric major histocompatibility complex class II I-A^k·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 V and V domains are connected by a flexible peptide linker. Purified and refolded D10 scTCR bound to SEC2 and murine major histocompatibility complex class II I-A^k·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 -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.

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