Soluble HIV-specific T cell receptor: expression, purification and analysis of the specificity.
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 alpha and beta chains under an inducible promoter. Both chains were labeled with two different tags: a (His)(6) was introduced at the C-terminal end of alpha chain, while beta chain was terminated by c-myc. Since an isolated alpha chain is unstable unless it is associated with a beta chain, this design permits rapid separation of alpha,beta-heterodimer from unpaired beta chain in a single step of Ni-NTA Agarose chromatography yielding 90% pure alpha,beta-TCR. Introduction of the c-myc epitope to the beta 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.[1]References
- Soluble HIV-specific T cell receptor: expression, purification and analysis of the specificity. Anikeeva, N., Lebedeva, T., Sumaroka, M., Kalams, S.A., Sykulev, Y. J. Immunol. Methods (2003) [Pubmed]
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