Monitoring protein-protein interactions using split synthetic renilla luciferase protein-fragment-assisted complementation
Monitoring protein-protein interactions using split synthetic renilla luciferase protein-fragment-assisted complementation
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DOI：
10.1021/ac020731c
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发表时间：
2003-04-01
影响因子：
7.4
通讯作者：
Gambhir, SS
中科院分区：
文献类型：
--
作者：
Paulmurugan, R;Gambhir, SS
在这项研究中，我们开发了一种诱导型合成海肾荧光素酶蛋白片段辅助互补的生物发光测定法，以定量测量哺乳动物细胞中的实时蛋白质-蛋白质相互作用。我们确定了合适的位点来生成蛋白质的 N 和 C 部分片段，通过互补产生显着恢复的活性。我们利用瞬时转染研究在五种不同的细胞系中验证了由两种强相互作用蛋白 MyoD 和 Id 的相互作用驱动的分裂合成海肾荧光素酶蛋白的基于互补的激活。该系统的表达水平还受到肿瘤坏死因子α通过用于驱动合成海肾N部分表达的NFκB启动子/增强子元件的调节。荧光素酶报告基因。这个新系统应该有助于研究蛋白质-蛋白质相互作用，并且当与其他分裂报告基因（例如分裂萤火虫荧光素酶）一起使用时，应该有助于监测细胞内网络的不同组成部分。
In this study we developed an inducible synthetic renilla luciferase protein-fragment-assisted complementation-based bioluminescence assay to quantitatively measure real time protein-protein interactions in mammalian cells. We identified suitable sites to generate fragments of N and C portions of the protein that yield significant recovered activity through complementation. We validate complementation-based activation of split synthetic renilla luciferase protein driven by the interaction of two strongly interacting proteins, MyoD and Id, in five different cell lines utilizing transient transfection studies. The expression level of the system was also modulated by tumor necrosis factor alpha through NFkappaB-promoter/enhancer elements used to drive expression of the N portion of synthetic renilla. luciferase reporter gene. This new system should help in studying protein-protein interactions and when used with other split reporters (e.g., split firefly luciferase) should help to monitor different components of an intracellular network.