Development of Bimolecular Fluorescence Complementation Using Dronpa for Visualization of Protein-Protein Interactions in Cells
Development of Bimolecular Fluorescence Complementation Using Dronpa for Visualization of Protein-Protein Interactions in Cells
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DOI：
10.1007/s11307-010-0312-2
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发表时间：
2010-10-01
影响因子：
3.1
通讯作者：
Han, Ye Sun
中科院分区：
文献类型：
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作者：
Lee, You Ri;Park, Jong-Hwa;Han, Ye Sun
Dronpa是一种新型的具有可逆光开关活性和快速响应光的荧光蛋白，我们开发了一种双分子荧光互补（BiFC）策略来监测细胞中蛋白质-蛋白质相互作用，Dronpa在Glu 164残基处被切割，产生两个Dronpa片段[Dronpa N-末端：DN（Met 1-Glu 164），Dronpa C-末端：DC（Gly 165-Lys 224）]。DN或DC分别与hHus 1的C端或hRad 1的N端融合。引入柔性接头[（GGGGS）x2]以通过hHus 1-hRad 1相互作用增强Dronpa互补。此外，我们开发了表达载体来可视化hMYH和hHus 1之间的相互作用。将hMYH和hHus 1编码区的基因片段分别与DN和DC编码区的N端或C端融合，仅在hHus 1-LDN和DCL-hRad 1共转染的HEK 293细胞中观察到互补的Dronpa荧光，而单独转染hHus 1-LDN或DCL-hRad 1表达载体的HEK 293细胞中未观察到互补的Dronpa荧光。使用抗c-myc或抗flag的免疫沉淀样品的Western印迹分析表明，DN融合的hHus 1与DC融合的hRad 1相互作用。在用hMYH-LDN和DCL-hHus 1表达载体或hMYH-LDN和hHus 1-LDC表达载体共转染的细胞中也观察到互补的Dronpa荧光。此外，通过hMYH-LDN和DCL-hHus 1相互作用诱导的Dronpa互补物显示出与天然Dronpa几乎相同的光开关活性。这些结果表明，使用Dronpa的BiFC可以成功地用于研究活细胞中蛋白质-蛋白质相互作用。此外，补充的Dronpa具有可逆的光开关活性的事实表明，它可以用作跟踪蛋白质-蛋白质相互作用的工具。
We developed a bimolecular fluorescence complementation (BiFC) strategy using Dronpa, a new fluorescent protein with reversible photoswitching activity and fast responsibility to light, to monitor protein-protein interactions in cells.Dronpa was split at residue Glu164 in order to generate two Dronpa fragments [Dronpa N-terminal: DN (Met1-Glu164), Dronpa C-terminal: DC (Gly165-Lys224)]. DN or DC was separately fused with C terminus of hHus1 or N terminus of hRad1. Flexible linker [(GGGGS)x2] was introduced to enhance Dronpa complementation by hHus1-hRad1 interaction. Furthermore, we developed expression vectors to visualize the interaction between hMYH and hHus1. Gene fragments corresponding to the coding regions of hMYH and hHus1 were N-terminally or C-terminally fused with DN and DC coding region.Complemented Dronpa fluorescence was only observed in HEK293 cells cotransfected with hHus1-LDN and DCL-hRad1 expression vectors, but not with hHus1-LDN or DCL-hRad1 expression vector alone. Western blot analysis of immunoprecipitated samples using anti-c-myc or anti-flag showed that DN-fused hHus1 interacted with DC-fused hRad1. Complemented Dronpa fluorescence was also observed in cells cotransfected with hMYH-LDN and DCL-hHus1 expression vectors or hMYH-LDN and hHus1-LDC expression vectors. Furthermore, complemented Dronpa, induced by the interaction between hMYH-LDN and DCL-hHus1, showed almost identical photoswitching activity as that of native Dronpa.These results demonstrate that BiFC using Dronpa can be successfully used to investigate protein-protein interaction in live cells. Furthermore, the fact that complemented Dronpa has a reversible photoswitching activity suggests that it can be used as a tool for tracking protein-protein interaction.