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.

中文翻译：

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改善三方sfGFP系统荧光信号的策略。

双分子荧光互补（BiFC）是一种用于检测蛋白质间相互作用的流行方法。对于BiFC分析，通常将荧光蛋白分为两部分，并在目标融合蛋白之间相互作用后恢复荧光。作为BiFC的优雅扩展，已开发出具有背景荧光低和融合标签大小小的优点的三方超折叠绿色荧光蛋白（sfGFP）系统。但是，在某些情况下，三方体系显示出低荧光信号。为了解决这个问题，我们建议通过添加亲和力对来增加三方系统的两个部分G1–9和G11之间的亲和力。在测试的三个亲和力对中，LgBiT-HiBiT最大程度地提高了信号和信噪比（S / N）。更惊人的是 G11与G1–9的直接共价融合，将三方体系转换为新的二分体系，使信噪比从20提高至146，这优于在157/158或173/174处分裂的sfGFP体系。我们的结果表明，sfGFP的第10条β链具有较低的亲和力和良好的回收效率，可以构建一个健壮的BiFC系统，并且该概念可能会应用于具有类似结构的其他荧光蛋白，以构建新的BiFC系统。