Skip to main content Accessibility help
×
Home

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

  • BENJAMIN E. WILLCOX (a1), GEORGE F. GAO (a1), JESSICA R. WYER (a1), CHRISTOPHER A. O'CALLAGHAN (a1), JONATHAN M. BOULTER (a2), E. YVONNE JONES (a2) (a3), P. ANTON VAN DER MERWE (a4), JOHN I. BELL (a1) (a2) and BENT K. JAKOBSEN (a1)...

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.

Copyright

Corresponding author

Reprint requests to: Bent K. Jakobsen, MRC Human Immunology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom; e-mail: jakobsen@pinnacle.jr2.ox.ac.uk.

Keywords

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

  • BENJAMIN E. WILLCOX (a1), GEORGE F. GAO (a1), JESSICA R. WYER (a1), CHRISTOPHER A. O'CALLAGHAN (a1), JONATHAN M. BOULTER (a2), E. YVONNE JONES (a2) (a3), P. ANTON VAN DER MERWE (a4), JOHN I. BELL (a1) (a2) and BENT K. JAKOBSEN (a1)...

Metrics

Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed