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.