Studies On Purification And Identification Of Protein Complexes Of P38 MAPK With TAP Technique

Systematic study of components and dynamic process of signaling transduction pathway net,which also has been the focus of study of "mics" presently,is the key to understanding the regulation and function mechanism of its own.p38 mitogen-activated protein kinase(MAPK) signaling pathway net is in charge of growth,development,differentiation and apoptosis processes,study of the dynamic change of protein complexes composed of important molecules of which will help us to understand the molecular mechanism of its regulation in all-round way.Tandem affinity purification(TAP) is a new technology for study of protein complexes,whose biggest advantage compared with other technics of purification is it can enrich protein complexes close to the physiological level efficiently.Taking advantage of advanced experiences of study of protein complexes in signaling transduction pathway from abroad and using TAP-MS,we have undertaken the research on p38 MAPK signaling pathway net.First,we successfully improved tandem affinity purification system by means of introducing the strptavidin binding protein(SBP) and calmodulin binding protein (CBP) as TAP tags for researching of protein complexes.Bait protein tagged by SBP which is composed of 45 amino acids,can be purified by immobilized stretavidin and then eluted by biotin.In the purification process,because of no need tobacco etch virus(TEV) protease for digestion,purified protein complexes will avoid being contaminated by macromolecular substance.Another advantage of SBP is that simple spatial structure efficiently reduced the influence to exact folding of bait protein and formation of the bait protein complex.The best advantage of TAP system is applied widely to the study of high eukaryotic cell protein complexes.We introduced the stable cell line selection system into TAP system by means of reconstruction of expression plasmid and construction of stable human embryonic kidney(HEK) 293 cell line capable of expressing p38 tagged with SBP and CBP.As a result,we can control the expression level of the bait close to the native physiological level,then purify the protein complex in physiological condition.In that way,components of the protein complex are more trustworthy.The principle of TAP is the sequential utilization of two tags for affinity-based purification.Because of the special structure of TAP tag,multi-step purification procedure is essential and beneficial for efficient reduction of unspecific proteins, which tend to result in the loss of associated proteins.So the key technology to purify genuine interaction partners of bait is using stringent purification conditions.After pre-experiment attempted,we optimized the protocol of TAP system for future research.SDS-PAGE is common separation technology for the protein complexes purified by TAP system.We applied advanced two-dimensional difference gel electrophoresis (2D-DIGE) to separate p38 protein complexes.DeCyder 2D analysis software found 35 different protein points between control group and treatment group,then dag out 14 protein points for identification by mass spectrometry(MS).Identification by mass spectrometry and database search are also the key steps to decide the reliability of protein complex,so set strict parameters and search standard library are very necessary.In this study,we got 6 candidate proteins of p38 protein complexes,actin reported in the literature has interaction with p38.these critical data reflects that identification results of mass spectrometry are certain credible.These proteins will help us to understand the complicated regulations for p38 functions.At the same time,TAP-MS strategy will help us to carry out the study of p38 MAPK signaling pathway. In short,we analyzed p38 protein complexes by comitant utilization TAP system and MS.Through the study,we draw the following conclusions:1.Through screening stable expression cell lines of HEK-pNTAP-p38 by G418,it is beneficial for reducing expression level of TAP-p38 fusion protein to native condition.As a result,we can purify credible interaction proteins of bait and reduce false positive interaction proteins.2.We improved purification efficiency of protein complex by optimizing TAP system condition which is favorable for affinity binding.That tend to find low-abundance proteins in the process of signaling transduction.3.After protein complexes were separated by 2D-DIGE,35 different protein points were found by DeCyder 2D analysis software,6 candidate proteins of p38 protein complex were identified by mass spectrometry from 14 different protein points.4.As candidate protein of p38 protein complex,actin is reported that has interaction with p38.Other proteins provide information for potential function of p38.