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Updated: Aug 1, 2026

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay
Published on: October 30, 2015
Heterodimeric, disulfide-linked alpha/beta T cell receptors in solution
1Department of Biology, Yale University School of Medicine, New Haven, CT 06510.
Researchers developed a soluble T cell receptor (TcR) by linking its extracellular domains to a Thy-1 anchor. This soluble TcR retains its native conformation, aiding structural and functional studies of TcR-ligand interactions.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Structural and functional analysis of T cell receptor (TcR)-ligand binding is crucial for understanding immune responses.
- The availability of intact, assembled TcRs in a soluble form would significantly advance research in this area.
Purpose of the Study:
- To produce an intact, assembled T cell receptor (TcR) in a soluble form for enhanced structural and functional analysis.
- To investigate the conformation and integrity of the generated soluble TcR.
Main Methods:
- Splicing extracellular domains of a TcR to the glycosyl phosphatidylinositol (GPI) membrane anchor sequences of Thy-1.
- Expressing the chimeric molecule in the absence of CD3.
- Cleaving the soluble TcR from the cell membrane using phosphatidylinositol-specific phospholipase C (PI-PLC).
- Assessing the paired alpha and beta chains using monoclonal antibodies (F23.1 and 1B2).
- Determining the molecular mass using SDS-PAGE under reducing and non-reducing conditions.
Main Results:
- A soluble alpha/beta TcR molecule was successfully produced.
- The soluble TcR was expressed on the cell surface in the absence of CD3 and could be cleaved by PI-PLC.
- The alpha and beta chains were confirmed to be paired in their native conformation, indicated by reactivity with specific monoclonal antibodies.
- SDS-PAGE analysis showed the molecule is a disulfide-linked dimer (95 kDa non-reducing) that dissociates into monomers (47 kDa reducing).
Conclusions:
- The study successfully generated an alpha/beta T cell receptor (TcR) in a soluble form.
- The produced soluble TcR retains its native conformation and disulfide linkage, making it suitable for structural and functional studies.
- This soluble TcR represents a valuable tool for advancing the understanding of TcR-ligand interactions.
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