Study On Preparation And Characterization Of Membrane-organizing Extension Spike Protein And Its Interaction With PIP2

The cortical cytoskeleton of eukaryotic cells provides structural support for the plasma membrane and participates in the dynamic process of a variety of cell life activities.Membrane-organizing extension spike protein（Moesin）plays a major role in the connection between the plasma membrane and the actin skeleton,the activation and migration of T cells,the regulation of the immune response of B cells,and the proliferation and migration of certain cancer cells.Under physiological conditions,Moesin is generally dormant.Previous studies have shown that the binding of phosphatidylinositol 4,5-bisphosphate（PIP2）and Moesin is the main way to activate Moesin,and the protein conformation changes significantly when activated.However,the thermodynamic parameters in the process of interaction between PIP2 and Moesin protein have not been clearly characterized,and it is also unclear how the protein conformation changes when Moesin is activated.In order to solve these problems,this subject uses isothermal titration calorimetry（ITC）and fluorescence resonance energy transfer（FRET）to study Moesin protein.The main contents are as follows:（1）The wild-type Moesin protein was prepared by the prokaryotic E.coli expression system,and the mutant of Moesin was successfully constructed.Firstly,the recombinant plasmid p ET28a-Moesin of wild-type Moesin was constructed,and the conditions for high-efficiency expression of Moesin protein were screened:Host bacteria:E.coli BL21,OD_600nm=0.6-0.8,induction temperature=18℃,inducer concentration=0.2 m M,induction time=8h.Subsequently,the target protein was extracted and purified by nickel ion affinity chromatography,gel chromatography and anion exchange chromatography.The purified protein was verified by SDS-PAGE and Western-blot,and the purity of the protein was≥95%.Finally,through site-directed mutagenesis and plasmid recombination,a Moesin mutant that can be used to construct the FRET system was successfully constructed.（2）The basic properties of wild-type Moesin and mutant proteins were characterized by circular dichroism spectrometer（CD）,and the mutant proteins were fluorescently labeled.The CD specrtra show that both wild-type Moesin and mutant proteins have the correct secondary structure.The system characterizes the thermodynamic stability of WT-Moesin protein and each mutant protein.The results of thermodynamic experiments show that the T_mof WT-Moeisn protein is 330.3±0.6 K,D_50%=3.52±0.2 M.Analyzing the Tm value and D50%value of other mutant proteins can be obtained:the removal of Cys at positions 117 and 284hardly affects the secondary structure of the protein,and the introduction of Cys at positions298/499 and 298/578 at the same time makes the stability of the protein slightly,the decrease of Cys at 499/578 at the same time makes the protein stability significantly reduced.In addition,the 117A-284A-499C-578C mutant protein was successfully labeled.（3）The thermodynamic parameters of the interaction between Moesin and PIP2 were characterized by isothermal titration calorimetry（ITC）.The binding ratio of wild-type Moesin protein and PIP2 is between 1:6,and the binding constant Ka=1.67?10⁷±0.33?10⁷ M^-1.The FRET phenomenon changed after the combination of PIP2 and Moesin,and a FRET research system was successfully constructed.