A novel orange-colored bimolecular fluorescence complementation (BiFC) assay using monomeric Kusabira-Orange protein
A novel orange-colored bimolecular fluorescence complementation (BiFC) assay using monomeric Kusabira-Orange protein
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DOI：
10.2144/btn-2017-0121
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发表时间：
2018-04-01
期刊：
影响因子：
2.7
通讯作者：
Kodama, Yutaka
中科院分区：
文献类型：
--
作者：
Fujii, Yuta;Yoshimura, Ayaka;Kodama, Yutaka
双分子荧光互补（BiFC）测定是一种可视化活细胞中蛋白质-蛋白质相互作用的工具。迄今为止，已经开发了许多类型的具有不同颜色的BiFC系统。除橙色外，可见光谱中的大多数颜色已用于BiFC测定。在这项研究中，我们利用来自石珊瑚真菌的Kusabira-Orange （KO）蛋白开发了一个橙色的bic系统。为了获得明亮的BiFC荧光，我们比较了两种KO单体变体（mKO1和mKO2）的荧光强度，发现mKO2比mKO1更亮。通过对互补效率和信噪比的比较分析，确定了基于mko2的bic的最佳分裂位点。由此产生的基于mko2的BiFC系统成功地证明了蛋白质二聚化在植物细胞中的模型实验。新型的基于mko2的bic系统将扩大多色bic分析的可能性。
The bimolecular fluorescence complementation (BiFC) assay was developed as a tool for the visualization of protein-protein interactions in living cells. To date, many types of BiFC systems with distinct colors have been developed. Most of the colors in the visible spectrum have been used in BiFC assays, with the exception of orange. In this study, we developed an orange-colored BiFC system using the Kusabira-Orange (KO) protein from the stony coral Fungia concinna. To obtain bright BiFC fluorescence, we compared fluorescence intensities of two monomeric KO variants (mKO1 and mKO2) and identified mKO2 as brighter than mKO1. The optimal split site for mKO2-based BiFC was defined by a comparative analysis of complementation efficiency and a signal-to-noise ratio. The resulting mKO2-based BiFC system successfully demonstrated protein dimerization in plant cells as a model experiment. The novel mKO2-based BiFC system will expand the possibility of multicolor BiFC analysis.