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Production of soluble αβ T-cell receptor heterodimers suitable for biophysical analysis of ligand binding

Published online by Cambridge University Press:  01 November 1999

BENJAMIN E. WILLCOX
Affiliation:
MRC Human Immunology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom
GEORGE F. GAO
Affiliation:
MRC Human Immunology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom
JESSICA R. WYER
Affiliation:
MRC Human Immunology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom
CHRISTOPHER A. O'CALLAGHAN
Affiliation:
MRC Human Immunology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom
JONATHAN M. BOULTER
Affiliation:
Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom
E. YVONNE JONES
Affiliation:
Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom Division of Structural Biology, The Wellcome Trust Center for Human Genetics, Roosevelt Drive, Oxford OX3 7BN, United Kingdom
P. ANTON VAN DER MERWE
Affiliation:
Sir William Dunn School of Pathology, University of Oxford, Oxford, OX1 3RE, United Kingdom
JOHN I. BELL
Affiliation:
MRC Human Immunology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom
BENT K. JAKOBSEN
Affiliation:
MRC Human Immunology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom
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Abstract

A method to produce αβ T-cell receptors (TCRs) in a soluble form suitable for biophysical analysis was devised involving in vitro refolding of a TCR fusion protein. Polypeptides corresponding to the variable and constant domains of each chain of a human and a murine receptor, fused to a coiled coil heterodimerization motif from either c-Jun (alpha) or v-Fos (beta), were overexpressed separately in Escherichia coli. Following recovery from inclusion bodies, the two chains of each receptor were denatured, and then refolded together in the presence of denaturants. For the human receptor, which is specific for the immunodominant influenza A HLA–A2-restricted matrix epitope (M58-66), a heterodimeric protein was purified in milligram yields and found to be homogeneous, monomeric, antibody-reactive, and stable at concentrations lower than 1 μM. Using similar procedures, analogous results were obtained with a murine receptor specific for an influenza nucleoprotein epitope (366–374) restricted by H2-Db. Production of these receptors has facilitated a detailed analysis of viral peptide–Major Histocompatibility Complex (peptide–MHC) engagement by the TCR using both surface plasmon resonance (SPR) and, in the case of the human TCR, isothermal titration calorimetry (ITC) (Willcox et al., 1999). The recombinant methods described should enable a wide range of TCR–peptide–MHC interactions to be studied and may also have implications for the production of other heterodimeric receptor molecules.

Type
Research Article
Information
Protein Science , Volume 8 , Issue 11 , November 1999 , pp. 2418 - 2423
Copyright
© 1999 The Protein Society

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Production of soluble αβ T-cell receptor heterodimers suitable for biophysical analysis of ligand binding
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