Strategies to improve the fluorescent signal of the tripartite sfGFP system.
Strategies to improve the fluorescent signal of the tripartite sfGFP system.
复制标题
改善三方 sfGFP 系统荧光信号的策略。
DOI：
10.1093/abbs/gmaa073
复制
发表时间：
2020
影响因子：
3.7
通讯作者：
Hu Jieli
中科院分区：
文献类型：
--
作者：
Shen Jing;Zhang Wenlu;Gan Chunyang;Wei Xiafei;Li Jie;Sun Yuxue;Yuan Yi;Cai Xuefei;Long Quanxin;Cui Jing;Guo Haitao;Huang Ailong;Hu Jieli
双分子荧光互补（BiFC）是一种常用的检测蛋白质-蛋白质相互作用的方法。对于BiFC测定，荧光蛋白通常被分成两部分，并且在目标融合蛋白之间的相互作用后恢复荧光。作为BiFC的一个优雅的扩展，已经开发了具有低背景荧光和小融合标签尺寸的优点的三重超折叠绿色荧光蛋白（sfGFP）系统。然而，在某些情况下，三组分体系表现出低荧光信号。为了解决这个问题，我们提出通过添加亲和对来增加三重系统的两个部分G1-9和G11之间的亲和性。在测试的三种亲和对中，LgBiT-HiBiT最大程度地改善了信号和信噪比（S/N）。更引人注目的是，G11与G1-9的直接共价融合将三部分系统转化为新的二部分系统，将S/N比从20提高到146，这上级于以157/158或173/174分裂的二部分sfGFP系统。我们的研究结果表明sfGFP的第10条β链具有低亲和力和良好的回收效率，可以构建稳定的BiFC系统，这一概念可以应用于其他具有类似结构的荧光蛋白，以构建新的BiFC系统。
Bimolecular fluorescence complementation (BiFC) is a popular method used to detect protein–protein interactions. For a BiFC assay, a fluorescent protein is usually split into two parts, and the fluorescence is recovered upon the interaction between the fused proteins of interest. As an elegant extension of BiFC, a tripartite superfold green fluorescent protein (sfGFP) system that has the advantages of low background fluorescence and small fusion tag size has been developed. However, the tripartite system exhibits a low fluorescence signal in some cases. To address this problem, we proposed to increase the affinity between the two parts, G1–9 and G11, of the tripartite system by adding affinity pairs. Among the three affinity pairs tested, LgBiT-HiBiT improved both the signal and signal-to-noise (S/N) ratio to the greatest extent. More strikingly, the direct covalent fusion of G11 to G1–9, which converted the tripartite system into a new bipartite system, enhanced the S/N ratio from 20 to 146, which is superior to the bipartite sfGFP system split at 157/158 or 173/174. Our results implied that the 10th β-strand of sfGFP has a low affinity and a good recovery efficiency to construct a robust BiFC system, and this concept might be applied to other fluorescent proteins with similar structure to construct new BiFC systems.