The Tandem Affinity Purification(TAP)is a powerful tool for the investigation of the protein-protein interactions under physiological or similar conditions. Many previous studies including our group's identified massive protein-protein interactions by manipulating the TAP system. However, almost all of these studies were focused on the interactions between the endocellular transcription factors, not so much on the extracellular ligand-transmembrane receptor mediated signal transduction process. This paper establishes an EX-TAP system for analyzing the extracellular ligand-transmembrane receptor mediated signal initial transduction process,constructs a prokaryotic recombinant protein expressing system and purifies a multi-tagged recombinant TNF-α from bacteria. The TNF-α is a pleiotropic pro-inflammatory factor which triggers the initial inflammation in vivo and is well defined in the past few decades, which is helpful for the identification of the feasibility of our EX-TAP system. By testing the phosphorylation of the inhibitor of the transcription factor κB, under the TNFR pathway, our recombinant TNF-α is determined biologically similar to the natural TNF-α and is functionally available to the EX-TAP system.

tandem affinity purification  /  protein purification  /  TNF-α/TNFR