State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, PR China.
State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, PR China.
Biochim Biophys Acta Gen Subj. 2022 Oct;1866(10):130201. doi: 10.1016/j.bbagen.2022.130201. Epub 2022 Jul 12.
Zinc(II) ions (Zn) play an essential role in living systems, with their delicate concentration balance differing among the various intracellular organelles. The spatiotemporal distribution and homeostasis of Zn can be monitored through photoluminescence imaging using zinc sensors. Among such biosensors, genetically encoded fluorescent sensor proteins are attractive tools owing to their subcellular localization advantage and high biocompatibility. However, the limited fluorescent properties of these proteins, such as their insufficient quantum yield and dynamic range, restrict their practical use. In this study, we developed an expression-screening-directed evolution system and used it to improve ZapCY1, a genetically encoded fluorescence resonance energy transfer (FRET) sensor. After four rounds of directed evolution, the FRET dynamic range of the modified sensor (designated ZapTV-EH) was increased by 1.5-1.7-fold. With its enhanced signal-to-noise ratio and ability to detect a wide Zn concentration range, ZapTV-EH proves to be a better visualization tool for monitoring Zn at the subcellular level. Combined with the simplified subcloning and expression steps and sufficient mutant libraries, this directed evolution system may provide a more simple and efficient way to develop and optimize genetically encoded FRET sensors through high-throughput screening.
锌离子（Zn）在生命系统中发挥着重要作用，其在各种细胞内细胞器中的浓度平衡存在差异。通过使用锌传感器进行荧光成像，可以监测 Zn 的时空分布和动态平衡。在这些生物传感器中，遗传编码的荧光传感器蛋白因其亚细胞定位优势和高生物相容性而成为有吸引力的工具。然而，这些蛋白的荧光特性有限，例如其量子产率和动态范围不足，限制了它们的实际应用。在本研究中，我们开发了一种表达筛选导向进化系统，并将其用于改进 ZapCY1，一种遗传编码的荧光共振能量转移（FRET）传感器。经过四轮定向进化，改良后的传感器（命名为 ZapTV-EH）的 FRET 动态范围增加了 1.5-1.7 倍。由于其增强的信噪比和检测宽 Zn 浓度范围的能力，ZapTV-EH 证明是一种更好的可视化工具，可用于在亚细胞水平监测 Zn。结合简化的亚克隆和表达步骤以及足够的突变文库，这种定向进化系统可能为通过高通量筛选开发和优化遗传编码 FRET 传感器提供更简单、更有效的方法。
