The Purification And Structural Analysis Of MIM-I-BAR Protein

As one of the important cell membrane regulatory proteins,Missing in Metastasis(MIM)is attracting more and more attention because of its abnormal expression in tumor.The most important structural feature of the MIM protein is the inclusion of the Inverse Bin-Amphiphysin-Rvs(I-BAR)domain,which allows dimerization of the MIM protein in vivo and related to the regulation of protein membrane,actin binding ability and Rac binding ability.Previous studies have shown that MIM dimers are also associated with endocytosis and the formation of filopodia,therefore the I-BAR domain and its dimerization are critical to the function of the MIM protein.The purified MIM-I-BAR protein can be used to study the biological macromolecule bound with MIM,to design and screen related drugs,and to study the effect of MIM-I-BAR protein on phospholipid membrane.Therefore,it is necessary to purify the high concentration MIM-I-BAR protein.AFM can achieve imaging of different samples at the nanometer scale and measure intermolecular interactions.Especially provide a lot of information for the study of protein,it can not only be used to measure the volume of protein and space structure,but also can be used to understand the interaction between protein and protein molecules,the specific interaction between DNA and protein molecules.In addition,the mild sample treatment of AFM avoids the denaturation of the sample during the observation.These advantages of AFM make it useful to guide the optimization of protein purification processes.In this study,the purification process of MIM-I-BAR protein was optimized by AFM,and the purity was 90%.The purified protein was detected by mass spectrometry and morphological observation.Specifically,the main work includes the following aspects:1)6 X His-tagged MIM-I-BAR protein was purified by Ni-NTA column.The solubility of the protein was successfully improved by optimizing the purification process,including changing the conditions of cell lysis and protein elution.The purity of MIM-I-BAR protein was up to 90%after purified by desalination and ultrafiltration step.2)The effects on the microenvironment of the protein after each purification steps were observed by atomic force microscopy,which was an innovative attempt.High-resolution atomic force microscopy(AFM)provided more visual images than traditional protein purification evaluation tools,such as SDS-PAGE and mass spectrometry,to ensure that we can observe the difference between each purification process.Combined with molecular dynamics simulation,the significance of the removal of high concentration imidazole in the process of protein purification was emphasized,which was instructive for.the subsequent purification of the protein.3)Finally,the purified MIM-I-BAR protein was subjected to secondary mass spectrometry and morphological observation.The results of secondary mass spectrometry were consistent with the MIM-I-BAR sequence in the Protein Data Bank.MIM-I-BAR protein with two different sizes was observed on the mica surface using atomic force microscopy.In combination with molecular dynamics simulations,these molecules are shown as MIM-I-BAR monomers and dimers.