Novel Methods For Protein Tyrosine Phosphorylation Analysis Of Tumor Cells Based On SH2 Superbinder

Objective:Aberrant protein phosphorylation plays a causal role in many diseases,particularly the tumor.Monitoring protein phosphorylation status in the tumor cells or tissues is vital for assessing the pathological phase,discovering the cancer biomarkers,and identifying the drug targets.Based on the "SH2 Superbiner" which shows high affinity for the protein tyrosine phosphorylation site,we developed a series of affinity reagents derived from SH2 superbinder for monitoring the protein tyrosine phosphorylation in different tumor cells as excellent substitutions of traditional anti-tyrosine phosphorylation monoclonal antibodies.Methods:The SH2 superbinder was transformed by gene recombination technology for some recombinant expression vectors,and the recombinant proteins were expressed,isolated and purified in E.coli.1.After fusion with a 6×His?histidine?tag to the N-terminus of SH2 protein,the His-SH2 superbinder protein was purified and used in the immunoprecipitation assay;2.Chemically combined the purified SH2 superbinder protein with HRP?horseradish peroxidase?and the conjugated HRP-SH2 superbinder reagent was used in the Western blot experiment;3.The EGFP-SH2 superbinder fusion protein through the same process of expression,separation and was used in the immunofluorescence experiment with fixed cell samples;4.The?Arg?9-EGFP-SH2 fusion protein was expressed and purified for intracellular tracking experiments of tyrosine phosphorylation proteins in live cells.Results:1.His-tagged SH2 superbinder protein can effectively separate and precipitate the phosphorylated protein complex through the combination of Beads and His-tag.2.The HRP-SH2 superbinder protein can effectively recognize the phosphoproteins imprinted on the membrane in the Western blot assay and can be directly visualized by chemiluminescence in one step.3.EGFP-SH2 superbinder fusion protein,which is used for immunofluorescence staining of fixed cells and can directly locate intracellular tyrosine phosphorylation proteins.4.?Arg?₉-EGFP-SH2 superbinder protein might efficiently enter into live cells and visually monitor their dynamic p-Tyr status under fluorescent microscope.Conclusion:The derivates of SH2 superbinder can efficiently and specifically recognize tyrosine phosphorylation sites in vitro.After comparison,a series of SH2 superbinder derived affinity reagents can reach or even surpass the effect of traditional tyrosine phosphorylation monoclonal antibodies in the analysis of protein tyrosine phosphorylation through different experiments.Our research provides a alternative method for the protein tyrosine phosphorylation associated analysis.