Preparation And Application Methodology Study Of Novel Transmembrane Protein-liposome Biochromatography

Transmembrane proteins belong to membrane proteins,which are embedded in lipid bilayers and exposed at both inner and outer surface of cell membranes.They play an important role in maintaining cell function.Transmembrane proteins are currently the most important drug targets,accounting for 70 % of all known drug targets at this stage.Therefore,drug discovery based on transmembrane proteins is an important way to innovate drug research and has been a hotspot in the field of pharmacology worldwide.This research focuses on transmembrane proteins and establishes a new transmembrane protein-liposome biochromatographic system,which is discussed from the following three aspects.1.Synthesis and methodological investigation of a new transmembrane proteinLiposome-silica gel stationary phaseThe structure of transmembrane proteins is complex and they have one or more hydrophobic regions so the purification,recombination and preservation of transmembrane proteins are inseparable from detergents.However,with the presence of detergents,the structure and function of proteins are destroyed,and the interaction between drugs and transmembrane proteins is quite different from the actual situation in vivo.In order to solve the problem,a series of new models and analytical techniques have been developed.The most representative one is proteoliposome reconstruction technology.The principle of this technology is to use various liposome to reconstruct artificial membranes to simulate the environment of cell membranes.Then transmembrane proteins are embedded on them to simulate the natural environment to characterize their interaction with drugs.However,proteoliposomes are not rigid enough to be used as chromatographic stationary phase.So they can not be applied to rapid and convenient liquid chromatography-mass spectrometry(LC-MS)technology.In order to solve this problem,referring to the technology of proteoliposome reconstruction and cell membrane chromatography,a new bacteriorhodopsin-liposome-silica gel stationary phase was constructed.The principle is to combine various liposomes into vesicles by membrane ultrasonic and hydration.Then bacteriorhodopsin was embedded on it.Next,the rhodopsin-liposome mixture was bond with silica gel by vacuum vortex.the physical parameters were investigated by particle size tester,scanning electron microscopy,and confocal microscopy.After the preparation of stationary phase,it was packed into the column by wet packing method.9-cis-retinal and dexamethasone was used as positive and negative drug to evaluate the effectiveness of the column.9-cis-retinal was injected for 7 consecutive days to investigate the life of the column.A series of experiments proved that the method successfully combines protein,liposome and silica gel to prepare a new chromatographic stationary phase.The validity and lifetime of this complex substance are verified by LC-MS,which lays a foundation for further popularization and application of this method.2.Preparation and application of comprehensive two-dimensional FZD4-reverse C18 column/TOF-MS chromatographic systemCell membrane chromatography(CMC)is a fast and sensitive method for the screening of active ingredients in complex systems of traditional Chinese medicine.However,although CMC has been developed for many years,there are still two shortcomings.Even highexpression cell lines which are transferred with specific protein expression vectors,also cannot eliminate the effects of other large amounts of necessary transmembrane proteins.Secondly,the target transmembrane proteins on cell membranes cannot be accurately quantified,and the abundance of the proteins is low.So the accuracy and sensitivity of interaction analysis will be affected.Therefore,a new two-dimensional transmembrane protein-liposome biochromatographic system has been developed in this chapter on the basis of the previous studies on cell membrane chromatography and the new stationary phases in the previous chapter.In this study,a two-dimensional FZD4-reverse C18 column/TOF-MS chromatographic system was established by frizzled family receptor 4(FZD4).FZD4,a seven-time transmembrane protein,belongs to the frizzled protein family.Numerous studies have shown that FZD4 is a potential target for colorectal adenocarcinoma,bladder cancer and other cancers by affecting the WNT/beta-catenin pathway.This comprehensive 2D chromatographic system was constructed to screen potential anticancer components of FZD4 protein in Pueraria lobata Ohwi and Scutellaria baicalensis Georgi.By comparing the results of mass spectrometry with components database and excluding the false positive results on blank control chromatography,five potential active ingredients in Pueraria lobata Ohwi and seven potential active ingredients in Scutellaria baicalensis Georgi were found in the comprehensive 2D FZD4 chromatographic system in this chapter.3.Validation of potential active components interacting with FZD4 from Pueraria lobata Ohwi and Scutellaria baicalensis GeorgiA 2D FZD4-reverse C18 column/TOF-MS chromatographic system has been established in the previous chapter andotential anti-cancer active ingredients which interacted with FZD4 have been screened.Some standards which can be purchased were obtained for further experiments.They were oroxylin A,wogonin,methyl palmitate,puerarin and 3’-hydroxypuerarin.To verify the anti-cancer activity of these components,human breast cancer cells(MCF7)were selected for a series of experiments.Firstly,the inhibition of cell proliferation experiment showed that oroxylin A,wogonin and puerarin had obvious killing effect on MCF7 cells.Secondly,Cell apoptotic experiments showed that oroxylin A,wogonin and puerarin kill cells through apoptotic pathway by flow cytometry.Then,western blotting results showed that wogonin and puerarin could significantly reduce the expression of FZD4 in MCF7 cells,while other compounds could not.Finally,the virtual docking of wogonin and puerarin with FZD4 protein was carried out by Discovery Studio 3.0 software.The results showed that wogonin and puerarin could be linked to the amino acid in binding site through hydrogen bond.