Characterizing the functionality of recombinant T-cell receptors in vitro: a pMHC tetramer based approach.
The very low affinity of the T-cell receptor (TCR) for the peptide-major histocompatibility complex (pMHC) has made it very challenging to design assays for testing the functionality of these molecules on small scales, which in turn has severely hampered the progress in developing expression and refolding methodologies for the TCR. We have now developed an ELISA assay for detecting pMHC binding to functional recombinant TCRs. It uses tetramers of biotinylated pMHCs bound to a neutravidin-horseradish peroxidase conjugate and detects the presence of functional TCR, bound in a productive orientation to an immobilized anti-Cbeta antibody. Specificity can be stringently demonstrated by inhibition with monomeric pMHCs. The assay is very sensitive and specific, and requires only very small amounts of protein. It has allowed us to study the unstable recombinant TCR P14, which we expressed and refolded from Escherichia coli. The TCR P14 is directed against the most abundant epitope of LCMV. We have confirmed the specificity of the interaction by BIAcore, and were able to determine the dissociation constant of the interaction of the P14 TCR and of the gp33-pMHC as 6 microM. This affinity ranks it among the tighter ones of TCR-pMHC interactions, and unusually low affinity thus does not seem to be the cause of the modest protective power of these T-cells, compared to others elicited in the anti-LCMV response. This strategy of multimerizing one partner and immobilizing the other in both a native form and productive orientation should be generally useful for characterizing the weak interactions of cell-surface molecules.
Tissot
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AC
Biochemisches Institut, Universität Zürich, Winterthurstrasse 190, CH-8057, Zürich, Switzerland.
Pecorari
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F
Plückthun
A
A
D000595:Amino Acid Sequence; D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000706:statistics & numerical data; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008217:Lymphocytic choriomeningitis virus / Q000276:immunology; D008285:Major Histocompatibility Complex; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity
D010455:Peptides; D011948:Receptors, Antigen, T-Cell; D011994:Recombinant Proteins