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.
KONG Chao
,
LI Dan
,
YUAN Xiaojun
,
LI Bin
. Purification and functional analysis of human recombinant tumor necrosis factor-α[J]. Science & Technology Review, 2017
, 35(8)
: 70
-74
.
DOI: 10.3981/j.issn.1000-7857.2017.08.008
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