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Production of recombinant TRAIL and TRAIL receptor: Fc chimeric proteins.
Methods in Enzymology
The tumor necrosis factor (TNF)/TNF receptor (TNFR) families of ligands and receptors are implicated in a variety of physiological and pathological processes and regulate cellular functions as diverse as proliferation, differentiation, and death. Recombinant forms of these ligands and receptors can act to agonize or antagonize these functions and are therefore useful for laboratory studies and may have clinical applications. A protocol is presented for the expression and purification of dimeric soluble receptors fused to the Fc portion of human IgG1 and of soluble, N-terminally Flag-tagged ligands. Soluble recombinant proteins are easier to handle than membrane-bound proteins and the use of tags greatly facilitates their detection and purification. In addition, some tags may provide enhanced biological activity to the recombinant proteins (mainly by oligomerization and stabilization effects) and facilitate their functional characterization. Expression in bacterial (for selected ligands) and eukaryotic expression systems (for ligands and receptors) was performed using M15 pREP4 bacteria and human embryonic kidney 293 cells, respectively. The yield of purified protein is about 1 mg/liter for the mammalian expression system and several milligrams per liter for the bacterial expression system. Protocols are given for a specific ligand-receptor pair, namely TRAIL (Apo-2L) and TRAIL receptor 2 (DR5), but can be applied to other ligands and receptors of the TNF family.
Amino Acid Sequence, Animals, Apoptosis, Apoptosis Regulatory Proteins, Base Sequence, Cloning, Molecular/methods, GPI-Linked Proteins, Genetic Techniques, Humans, Immunoglobulin Fc Fragments, Immunoglobulin G, Ligands, Mammals, Membrane Glycoproteins/biosynthesis, Membrane Glycoproteins/genetics, Molecular Sequence Data, Plasmids, Polymerase Chain Reaction/methods, Promoter Regions, Genetic, Receptors, Tumor Necrosis Factor/biosynthesis, Receptors, Tumor Necrosis Factor/genetics, Recombinant Fusion Proteins/biosynthesis, Recombinant Fusion Proteins/metabolism, TNF-Related Apoptosis-Inducing Ligand, Tumor Necrosis Factor Decoy Receptors, Tumor Necrosis Factor-alpha/biosynthesis, Tumor Necrosis Factor-alpha/genetics
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