Production of soluble alphabeta T-cell receptor heterodimers suitable for biophysical analysis of ligand binding.
A method to produce alphabeta 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 microM. 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.
Willcox
B E
BE
MRC Human Immunology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom.
Gao
G F
GF
Wyer
J R
JR
O'Callaghan
C A
CA
Boulter
J M
JM
Jones
E Y
EY
van der Merwe
P A
PA
Bell
J I
JI
Jakobsen
B K
BK
D000595:Amino Acid Sequence; D001665:Binding Sites; D001703:Biophysics / Q000379:methods; D019281:Dimerization; D015789:HLA-A2 Antigen / Q000737:chemistry; D006801:Humans; D016350:Leucine Zippers; D008024:Ligands; D008285:Major Histocompatibility Complex; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016761:Oncogene Proteins v-fos / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D016755:Proto-Oncogene Proteins c-jun / Q000737:chemistry; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000737:chemistry* / Q000378:metabolism; D012995:Solubility; D020349:Surface Plasmon Resonance
D015789:HLA-A2 Antigen; D008024:Ligands; D016761:Oncogene Proteins v-fos; D016755:Proto-Oncogene Proteins c-jun; D016693:Receptors, Antigen, T-Cell, alpha-beta