Production of soluble alpha beta T-cell receptor heterodimers suitable forbiophysical analysis of ligand binding

A method to produce alpha beta T-cell receptors (TCRs) in a soluble form su itable for biophysical analysis was devised involving in vitro refolding of a TCR fusion protein. Polypeptides corresponding to the variable and const ant domains of each chain of a human and a murine receptor, fused to a coil ed 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 matr ix epitope (M58-66), a heterodimeric protein was purified in milligram yiel ds and found to be homogeneous, monomeric, antibody-reactive, and stable at concentrations lower than 1 mu M. Using similar procedures, analogous resu lts were obtained with a murine receptor specific for an influenza nucleopr otein epitope (366-374) restricted by H2-D-b. Production of these receptors has facilitated a detailed analysis of viral peptide-Major Histocampabilit y Complex (peptide-MHC) engagement by the TCR using both surface plasmon re sonance (SPR) and, in the case of the human TCR, isothermal titration calor imetry (ITC) (Willcox ct al., 1999). The recombinant methods described shou ld enable a wide range of TCR-peptide-MHC interactions to be studied and ma y also have implications for the production of other heterodimeric receptor molecules.