Soluble HIV-specific T cell receptor: expression, purification and analysis of the specificity

J Immunol Methods. 2003 Jun 1;277(1-2):75-86. doi: 10.1016/s0022-1759(03)00179-0.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Biosensing Techniques
  • Chromatography, Agarose
  • Drosophila / genetics
  • Enzyme-Linked Immunosorbent Assay
  • Epitopes / immunology
  • Genes, gag / immunology
  • HIV / genetics
  • HIV / immunology*
  • HLA-A2 Antigen / immunology
  • HLA-A2 Antigen / metabolism
  • Humans
  • Molecular Sequence Data
  • Receptors, Antigen, T-Cell, alpha-beta / genetics
  • Receptors, Antigen, T-Cell, alpha-beta / immunology*
  • Receptors, Antigen, T-Cell, alpha-beta / isolation & purification*
  • Receptors, Antigen, T-Cell, alpha-beta / metabolism
  • Recombinant Proteins / genetics
  • Recombinant Proteins / immunology

Substances

  • Epitopes
  • HLA-A2 Antigen
  • Receptors, Antigen, T-Cell, alpha-beta
  • Recombinant Proteins