蛋白质通量化定位体系的建立及初步应用

目的:对于蛋白质功能而言,蛋白质定位与蛋白质的表达和修饰等同等重要。传统的蛋白质定位一直沿用单个基因、逐个的研究方法,本实验拟建立一种通量蛋白质定位研究体系。方法:采用并优化了细胞微阵列技术,结合绿色荧光蛋白(GFP)标签、激光扫描共聚焦显微镜及反转染技术,用于大规模蛋白质定位研究。结果:初步建立的蛋白质定位微阵列包含107个GFP标记的cDNA表达载体,分别编码107个重要细胞信号传导通路的蛋白质,并与定位数据库中的已知结果进行了比对;对该系统的有效性进行了验证评价。结论:本定位系统可有效地用于通量化蛋白质定位研究,并可以发展用于蛋白质相互作用、泛素-蛋白酶体通路底物筛选等进一步的功能研究。

High-Throughput Identification of Protein Localization on Cell Arrays

Objective: Protein localization is equally important to protein expression and modification for functional proteomic studies,and it's necessary to obtain large scale information of these.To develop a cell microarray-based technology for highly parallel analysis of protein localization.Methods: Specifically,107 target genes were first construct into pEGFP-N1 vector separately,then plasmids in a transfection matrix were arrayed on treated glass slides,overlaid with a mono-layer of adherent cells,incubated to allow reverse transfection and GFP tagged protein expressed specifically at distinct spots,finally,protein localization microarrays imaged and analyzed based on auto-fluorescence or immunofluorescence by laser scanning confocal microscopy at a single cell level.Results: A high-throughput protein localization system for subcellular location analyses and dynamic observation of thousands of protein was established,which integrated microarray technology,solidity reverse transfection,laser scanning confocal microscopy and automatical data archiving.In addition,localization of 107 proteins and validate in database was studied.Conclusion: This protein-cell microarray platform,together with ongoing efforts to develop large-scale protein subcellular localization libraries,should facilitate proteome-scale cell-based analysis of protein function.Furthermore,proteome-wide application of the protein localization in combination with the cell array technology has more challenging application,such as accelerating elucidation of cell signaling pathways or mechanisms of programmed cell death and so on.