We have produced soluble T cell receptor (TCR) derived from a human CD8 + cytotoxic T lymphocyte (CTL) clone D3 that recognizes the immunodominant HIV Gag peptide SLYNTVATL (SL9) in association with major histocompatibility complex (MHC) class I protein HLA-A2. Drosophila Schneider cells (S2) were used to express genes coding the TCR α and β chains under an inducible promoter. Both chains were labeled with two different tags: a (His) 6 was introduced at the C-terminal end of α chain, while β chain was terminated by c-myc. Since an isolated α chain is unstable unless it is associated with a β chain, this design permits rapid separation of α,β-heterodimer from unpaired β chain in a single step of Ni-NTA Agarose chromatography yielding 90% pure α,β-TCR. Introduction of the c-myc epitope to the β chain allows capture of soluble D3 from the culture supernatant by immobilized anti-c-myc antibody, without the need for receptor purification. The TCR specificity was then examined by analyzing the binding of peptide-HLA-A2/tetramer in an ELISA assay. Using this assay, we have also evaluated the binding of monomeric SL9-HLA-A2 complex to the immobilized D3 TCR and determined that the affinity measurement of the D3-SL9-HLA-A2 reaction is similar to that obtained by a biosensor instrument. We propose that the approach described here is generally useful for purification of other soluble TCRs and will allow rapid analysis of their specificity.
Financed by the National Centre for Research and Development under grant No. SP/I/1/77065/10 by the strategic scientific research and experimental development program:
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