New methods for delivering proteins into the cytosol of mammalian cells are being reported at a rapid pace. Differentiating between these methods in a quantitative manner is difficult, however, as most assays for evaluating cytosolic protein delivery are qualitative and indirect and thus often misleading. Here we make use of fluorescence correlation spectroscopy (FCS) to determine with precision and accuracy the relative efficiencies with which seven different previously reported “cell-penetrating peptides” (CPPs) transport a model protein cargo—the self-labeling enzyme SNAP-tag—beyond endosomal membranes and into the cytosol. Using FCS, we discovered that the miniature protein ZF5.3 is an exceptional vehicle for delivering SNAP-tag to the cytosol. When delivered by ZF5.3, SNAP-tag can achieve a cytosolic concentration as high as 250 nM, generally at least 2-fold and as much as 6-fold higher than any other CPP evaluated. Additionally, we show that ZF5.3 can be fused to a second enzyme cargo—the engineered peroxidase APEX2—and reliably delivers the active enzyme to the cell interior. As FCS allows one to realistically assess the relative merits of protein transduction domains, we anticipate that it will greatly accelerate the identification, evaluation, and optimization of strategies to deliver large, intact proteins to intracellular locales.

中文翻译：

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荧光相关光谱揭示了细胞渗透性微型蛋白质对蛋白质货物的有效胞质递送

将蛋白质输送到哺乳动物细胞胞浆中的新方法正在快速报道。然而，以定量方式区分这些方法很困难，因为大多数评估胞质蛋白递送的测定都是定性和间接的，因此经常产生误导。在这里，我们利用荧光相关光谱 (FCS) 来精确确定先前报道的七种不同的“细胞穿透肽”(CPP) 运输模型蛋白质货物（自标记酶 SNAP 标签）的相对效率。超出内体膜并进入细胞质。使用 FCS，我们发现微型蛋白 ZF5.3 是将 SNAP 标签传递到细胞质的特殊载体。当通过 ZF5.3 传递时，SNAP-tag 的胞质浓度可高达 250 nM，通常比任何其他评估的 CPP 高至少 2 倍、最多 6 倍。此外，我们表明 ZF5.3 可以与第二种酶货物（工程过氧化物酶 APEX2）融合，并将活性酶可靠地递送到细胞内部。由于 FCS 允许人们现实地评估蛋白质转导域的相对优点，我们预计它将大大加速将大的、完整的蛋白质递送到细胞内区域的策略的识别、评估和优化。