Recombinant TRAIL and TRAIL receptor analysis.
Death receptors are a subgroup of the tumor necrosis factor receptor superfamily (TNFRSF) and mediate activation of what is widely known as the "extrinsic" apoptosis pathway. TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is one of the most recent death receptor ligands identified. The TRAIL receptor family consists of four distinct membrane-bound receptors, named TRAIL-R1 to -R4. TRAIL-R1 and TRAIL-R2 belong to the "death receptor" subfamily of the TNFRSF. Unlike other death receptor ligands, such as FasL/CD95L and TNF, TRAIL seems to display selective toxicity by killing tumor and transformed, but not normal, cells. Importantly, there also seems 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 labeling of recombinant TRAIL with either biotin or fluorochromes. These recombinant TRAIL preparations are then used to study various aspects of TRAIL signaling 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 signaling.
Harper
Nicholas
N
MRC Toxicology Unit, University of Leicester, Leicester, LE1 9HN, UK.
MacFarlane
Marion
M
D017304:Annexin A5 / Q000502:physiology; D000906:Antibodies / Q000378:metabolism; D017209:Apoptosis / Q000502:physiology; D019921:Biotinylation / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000032:analysis* / Q000276:immunology; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000032:analysis* / Q000096:biosynthesis
D017304:Annexin A5; D000906:Antibodies; D005456:Fluorescent Dyes; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D011994:Recombinant Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand