Design, engineering and production of functional single-chain T cell receptor ligands

Major histocompatibility complex (MHC) class II molecules are membrane-anchored heterodimers on the surface of antigen presenting cells (APCs) that bind the T cell receptor, initiating a cascade of interactions that results in antigen-specific activation of clonal populations of T cells. The peptide binding/T cell recognition domains of rat MHC class II (alpha-1 and beta-1 domains) were expressed as a single exon for structural and functional characterization. These recombinant single-chain T cell receptor ligands (termed `β1α1' molecules) of approximately 200 amino acid residues were designed using the structural backbone of MHC class II molecules as template, and have been produced in Escherichia coli with and without N-terminal extensions containing antigenic peptides. Structural characterization using circular dichroism predicted that these molecules retained the antiparallel β-sheet platform and antiparallel α-helices observed in the native MHC class II heterodimer. The proteins exhibited a cooperative two-state thermal folding–unfolding transition. β1α1 molecules with a covalently linked MBP-72–89 peptide showed increased stability to thermal unfolding relative to the empty β1α1 molecules. This new class of small soluble polypeptide provides a template for designing and refining human homologues useful in detecting and regulating pathogenic T cells.