Recombinant TRAIL and TRAIL Receptor Analysis.

Death receptors are a subgroup of the Tumour Necrosis Factor Receptor Superfamily (TNFRSF) and mediate activation of what is widely known as the ‘extrinsic’ apoptosis pathway. TRAIL (Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand) is one of the most recent death receptor ligands identified. The TRAIL receptor family consists of four distinct membranebound receptors, named TRAIL-R1 to -R4. TRAIL-R1 and TRAIL-R2 belong to the ‘death receptor’ subfamily of the Tumour Necrosis Factor Receptor Superfamily (TNFRSF). Unlike other death receptor ligands, such as FasL/CD95L and TNF, TRAIL appears to display selective toxicity by killing tumour and transformed but not normal cells. Importantly, there also appears to be a complete lack of apparent toxicity, specifically hepatotoxicity, when TRAIL is used in vivo. Taken together, these observations led to TRAIL being proposed as a potential anti-tumor therapeutic, thus explaining the intense activity surrounding TRAIL and TRAIL receptor research over the past few years. This chapter describes a number of methods for the production of recombinant TRAIL in E.coli followed by labelling of recombinant TRAIL with either biotin or fluorochromes. These recombinant TRAIL variants are then employed to study various aspects of TRAIL signalling from cell surface receptor levels to the composition of death receptor complexes. This combination of direct binding and functional analysis provides a very powerful approach to aid in further characterization of TRAIL/TRAIL receptor regulation and signalling.