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.
Anikeeva
Nadja
N
Department of Microbiology and Immunology and Kimmel Cancer Institute, BLSB 650, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Lebedeva
Tatiana
T
Sumaroka
Marina
M
Kalams
Spyros A
SA
Sykulev
Yuri
Y
D000595:Amino Acid Sequence; D000818:Animals; D015374:Biosensing Techniques; D002847:Chromatography, Agarose; D004330:Drosophila / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D015750:Genes, gag / Q000276:immunology; D006678:HIV / Q000235:genetics / Q000276:immunology*; D015789:HLA-A2 Antigen / Q000276:immunology / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology
D000939:Epitopes; D015789:HLA-A2 Antigen; D016693:Receptors, Antigen, T-Cell, alpha-beta; D011994:Recombinant Proteins