Tens of thousands of protein-protein interactions (PPIs) have been found in human cells and many of these macromolecular partnerships could determine the cell growth and death. Thus there is a need to develop the meth...Tens of thousands of protein-protein interactions (PPIs) have been found in human cells and many of these macromolecular partnerships could determine the cell growth and death. Thus there is a need to develop the methods to catalogue these macromolecules by detecting their interactions, modifications, and cellular locations. It will be helpful for scientists to compare the difference between a diseased cellular state and its normal state and to find the potential therapy treatment to intervene this status. One technology called split-protein reassembly or protein fragment complementation has been developed in the last two decades. This technology makes use of appropriate fragmentation of some protein reporters and the refolding of these reports could be detected by their function to confirm the interaction of interest. This system has been set up in cell-free systems, </span><i><span style="font-family:Verdana;">E.</span></i></span><i><span style="font-family:""> </span></i><i><span style="font-family:Verdana;">coli</span></i><span style="font-family:""><span style="font-family:Verdana;">, yeast, mammalian cells, plants and live animals. Herein, I present the development in fluorescence- and bioluminescence-based split-protein biosensors in both binary and ternary systems. In addition, some people developed the split-protein system by combining it with chemical inducer of dimerization strategy (CID). This has been applied for identifying the enzyme inhibitors and regulating the activity of protein kinases and phosphatases. With effort from many laboratories from the world, a variety of split-protein systems have been developed for studying the PPI </span><i><span style="font-family:Verdana;">in vitro</span></i><span style="font-family:Verdana;"> and </span><i><span style="font-family:Verdana;">in vivo</span></i><span style="font-family:Verdana;">, monitoring the biological process, and controlling the activity of the enzyme of interest.展开更多
【背景】布鲁氏菌(Brucella)作为一种兼性胞内寄生菌,可通过分泌效应蛋白修饰宿主细胞多重信号通路帮助其完成胞内增殖。因此,分泌效应蛋白的鉴定对于阐明布鲁氏菌致病机制至关重要。细菌分泌效应蛋白的鉴定方法通常有融合标签法和酶标...【背景】布鲁氏菌(Brucella)作为一种兼性胞内寄生菌,可通过分泌效应蛋白修饰宿主细胞多重信号通路帮助其完成胞内增殖。因此,分泌效应蛋白的鉴定对于阐明布鲁氏菌致病机制至关重要。细菌分泌效应蛋白的鉴定方法通常有融合标签法和酶标记技术,但其操作步骤烦琐、成本昂贵。【目的】基于双分子荧光互补技术开发一种简单便捷的质粒系统,用来鉴定布鲁氏菌的分泌效应蛋白。【方法】基于分裂绿色荧光蛋白(split green fluorescent protein,split-GFP)技术,首先,构建真核表达质粒pMAX-sfGFP_(1-10)-IRES-NLS-tagBFP,该质粒能在转染的细胞中表达sfGFP_(1-10)蛋白片段,同时具有核定位信号的BFP蛋白能指示转染成功的细胞;之后,构建能在布鲁氏菌中融合表达4×GFP_(11)蛋白的pMCRt-mCherry-P31-4×GFP_(11)-Flag质粒。最后,通过布鲁氏菌分泌效应蛋白BspA和BPE123以及阴性蛋白GST验证split-GFP系统的可行性。【结果】荧光显微镜观察转染pMAX-sfGFP_(1-10)-IRES-NLS-tagBFP质粒的HeLa细胞,表明tagBFP和sfGFP_(1-10)蛋白成功表达;蛋白免疫印迹试验证明布鲁氏菌BspA、BPE123及GST成功与4×GFP_(11)-Flag融合表达;细胞感染和荧光显微镜观察结果表明split-GFP能成功指示BspA和BPE123的分泌活性,而阴性分泌蛋白GST无分泌活性。【结论】本研究基于split-GFP技术构建的双质粒系统能成功验证布鲁氏菌效应蛋白的分泌活性,为布鲁氏菌分泌蛋白的鉴定提供了一种新的工具。展开更多
文摘Tens of thousands of protein-protein interactions (PPIs) have been found in human cells and many of these macromolecular partnerships could determine the cell growth and death. Thus there is a need to develop the methods to catalogue these macromolecules by detecting their interactions, modifications, and cellular locations. It will be helpful for scientists to compare the difference between a diseased cellular state and its normal state and to find the potential therapy treatment to intervene this status. One technology called split-protein reassembly or protein fragment complementation has been developed in the last two decades. This technology makes use of appropriate fragmentation of some protein reporters and the refolding of these reports could be detected by their function to confirm the interaction of interest. This system has been set up in cell-free systems, </span><i><span style="font-family:Verdana;">E.</span></i></span><i><span style="font-family:""> </span></i><i><span style="font-family:Verdana;">coli</span></i><span style="font-family:""><span style="font-family:Verdana;">, yeast, mammalian cells, plants and live animals. Herein, I present the development in fluorescence- and bioluminescence-based split-protein biosensors in both binary and ternary systems. In addition, some people developed the split-protein system by combining it with chemical inducer of dimerization strategy (CID). This has been applied for identifying the enzyme inhibitors and regulating the activity of protein kinases and phosphatases. With effort from many laboratories from the world, a variety of split-protein systems have been developed for studying the PPI </span><i><span style="font-family:Verdana;">in vitro</span></i><span style="font-family:Verdana;"> and </span><i><span style="font-family:Verdana;">in vivo</span></i><span style="font-family:Verdana;">, monitoring the biological process, and controlling the activity of the enzyme of interest.
文摘【背景】布鲁氏菌(Brucella)作为一种兼性胞内寄生菌,可通过分泌效应蛋白修饰宿主细胞多重信号通路帮助其完成胞内增殖。因此,分泌效应蛋白的鉴定对于阐明布鲁氏菌致病机制至关重要。细菌分泌效应蛋白的鉴定方法通常有融合标签法和酶标记技术,但其操作步骤烦琐、成本昂贵。【目的】基于双分子荧光互补技术开发一种简单便捷的质粒系统,用来鉴定布鲁氏菌的分泌效应蛋白。【方法】基于分裂绿色荧光蛋白(split green fluorescent protein,split-GFP)技术,首先,构建真核表达质粒pMAX-sfGFP_(1-10)-IRES-NLS-tagBFP,该质粒能在转染的细胞中表达sfGFP_(1-10)蛋白片段,同时具有核定位信号的BFP蛋白能指示转染成功的细胞;之后,构建能在布鲁氏菌中融合表达4×GFP_(11)蛋白的pMCRt-mCherry-P31-4×GFP_(11)-Flag质粒。最后,通过布鲁氏菌分泌效应蛋白BspA和BPE123以及阴性蛋白GST验证split-GFP系统的可行性。【结果】荧光显微镜观察转染pMAX-sfGFP_(1-10)-IRES-NLS-tagBFP质粒的HeLa细胞,表明tagBFP和sfGFP_(1-10)蛋白成功表达;蛋白免疫印迹试验证明布鲁氏菌BspA、BPE123及GST成功与4×GFP_(11)-Flag融合表达;细胞感染和荧光显微镜观察结果表明split-GFP能成功指示BspA和BPE123的分泌活性,而阴性分泌蛋白GST无分泌活性。【结论】本研究基于split-GFP技术构建的双质粒系统能成功验证布鲁氏菌效应蛋白的分泌活性,为布鲁氏菌分泌蛋白的鉴定提供了一种新的工具。
