Cytosolic protein delivery promises diverse applications from therapeutics, to genetic modification and precision research tools. To achieve effective cellular and subcellular delivery, approaches that allow protein visualization and accurate localization with greater sensitivity are essential. Fluorescently tagging proteins allows detection, tracking and visualization in cellulo. However, undesired consequences from fluorophores or fluorescent protein tags, such as nonspecific interactions and high background or perturbation to native protein's size and structure, are frequently observed, or more troublingly, overlooked. Distinguishing cytosolically released molecules from those that are endosomally entrapped upon cellular uptake is particularly challenging and is often complicated by the inherent pH‐sensitive and hydrophobic properties of the fluorophore. Monitoring localization is more complex in delivery of proteins with inherent protein‐modifying activities like proteases, transacetylases, kinases, etc. Proteases are among the toughest cargos due to their inherent propensity for self‐proteolysis. To implement a reliable, but functionally silent, tagging technology in a protease, we have developed a caspase‐3 variant tagged with the 11th strand of GFP that retains both enzymatic activity and structural characteristics of wild‐type caspase‐3. Only in the presence of cytosolic GFP strands 1–10 will the tagged caspase‐3 generate fluorescence to signal a non‐endosomal location. This methodology facilitates easy screening of cytosolic vs. endosomally‐entrapped proteins due to low probabilities for false positive results, and further, allows tracking of the resultant cargo's translocation. The development of this tagged casp‐3 cytosolic reporter lays the foundation to probe caspase therapeutic properties, charge–property relationships governing successful escape, and the precise number of caspases required for apoptotic cell death.

中文翻译：

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使用 split GFP 追踪外源细胞内 casp-3

胞质蛋白递送有望实现从治疗到基因修饰和精密研究工具的多种应用。为了实现有效的细胞和亚细胞递送，允许蛋白质可视化和具有更高灵敏度的准确定位的方法至关重要。荧光标记蛋白质可以在细胞中进行检测、跟踪和可视化。然而，荧光团或荧光蛋白标签带来的不良后果，例如非特异性相互作用和高背景或对天然蛋白质大小和结构的扰动，经常被观察到，或更令人不安的是，被忽视。区分细胞溶质释放的分子与细胞摄取时被内体捕获的分子特别具有挑战性，并且通常由于荧光团固有的 pH 敏感性和疏水性而变得复杂。在具有固有蛋白质修饰活性（如蛋白酶、转乙酰酶、激酶等）的蛋白质递送中，监测定位更加复杂。蛋白酶由于其固有的自我蛋白水解倾向，是最难运输的货物之一。为了在蛋白酶中实施可靠但功能上沉默的标记技术，我们开发了一种用 GFP 第 11 链标记的 caspase-3 变体，它保留了野生型 caspase-3 的酶活性和结构特征。仅当存在胞质 GFP 链 1-10 时，标记的 caspase-3 才会产生荧光以指示非内体位置。由于假阳性结果的可能性较低，该方法有利于轻松筛选胞质蛋白与内体包埋蛋白，并且还可以跟踪所得货物的易位。这种标记的 casp-3 胞质报告基因的开发为探测 caspase 治疗特性、控制成功逃逸的电荷-特性关系以及细胞凋亡所需的 caspase 精确数量奠定了基础。