, 1996). These structures provided the first insight into T cell antigen recognition and revealed a number of important features of the interface between the TCR and pMHC. Ten years later, only 10 unique human TCR/pMHC complexes had been solved, as reviewed by Rudolph et al. (2006). In recent years, this number has increased to ~ 25 human TCR/pMHC complexes, but progress has still been relatively slow compared with the number

of antibody structures, or unligated pMHC structures that have been reported. This lack of structural information selleck screening library regarding human TCR/pMHC complexes has compromised the determination of a comprehensive and accepted set of rules that govern T cell antigen recognition and a number of conflicting theories still dominate the field (Bridgeman et al., 2012). Difficulties in generating sufficient quantities of soluble TCR and pMHC protein, and in producing high quality TCR/pMHC Selleckchem HKI 272 complex crystals, may explain the low number of these structures. Additionally, TCRs bind to pMHCs with relatively weak affinity (KD = 0.1–300 μM (Cole et al., 2007 and Bridgeman et al., 2012)), which may further impede their ability to form stable complexes for crystallization. A number of approaches have been proposed for the production of stable, soluble recombinant TCRs, including modification of the expression

vectors and optimization of culture conditions. To date, soluble Epothilone B (EPO906, Patupilone) TCRs have been generated using various eukaryotic expression systems such as: Drosophila melanogaster ( Garcia et al., 1996), myeloma cells ( Wang et al., 1998), Chinese hamster ovary cells ( Reiser et al., 2000) and Spodoptera frugiperda cells ( Hahn et al., 2005). However, prokaryotic expression as inclusion bodies using Escherichia coli

strains, followed by artificial refolding, remains the most popular and robust system because it produces high yields of homogenous protein ( Cole et al., 2007, Cole et al., 2008 and Cole et al., 2009). Additionally, four different TCR cloning methods have been designed to improve soluble TCR stability including: (1) expression of the variable domains only in a form of a single chain Fv fragment (scFv) ( Housset et al., 1997); (2) expression of TCR α and β chains carrying c-Jun (α) and c-Fos (β) leucine-zipper heterodimerization motifs at their carboxyl termini ( Garcia et al., 1996); (3) introduction of a carboxy-terminal flanking sequence to the full length V and C ectodomains to promote the formation of an interchain disulphide bridge ( Stewart-Jones et al., 2003); and, (4) introduction of a non-native disulphide bond into the interface between the TCR constant domains ( Boulter et al., 2003). The ‘Boulter-disulphide’ method has been the preferred choice in our laboratory.