Discovery and characterization of a new cell-penetrating protein.
Discovery and characterization of a new cell-penetrating protein.
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DOI：
10.1021/cb4004089
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发表时间：
2013-12-20
影响因子：
4
通讯作者：
Chen Z
中科院分区：
文献类型：
--
作者：
Simeon RL;Chamoun AM;McMillin T;Chen Z
我们描述了一种新的细胞穿透蛋白，B1，能够提供共轭蛋白质和核酸到哺乳动物细胞。B1是编码增强型绿色荧光蛋白（eGFP）的基因中单碱基移码的244个氨基酸产物。该分子具有43的净正电荷和1.5的非常高的荷质比。eGFP融合的B1有效地穿透粘附细胞和悬浮细胞，当暴露于低至1 μM的浓度时，>80%的细胞摄取蛋白质。发现该蛋白聚集在TZM-bl细胞的核旁区域。最重要的是，我们表明，B1不仅有利于细胞摄取，但允许生物分子货物到达生物相关的网站。例如，当暴露于低至2.5 μM的蛋白质浓度的B1标记的Cre重组酶时，幼仓鼠肾细胞经历DNA重组，表明功能性蛋白质货物的有效核递送。此外，B1将非共价结合的RNA和DNA穿过细胞膜递送至细胞翻译和转录机构可接近的胞质和核位点，如通过检测编码的报告功能所衡量的，其效率与市售阳离子脂质试剂相当。B1似乎利用细胞表面聚糖和多种竞争性内吞途径进入细胞并在细胞中运输。这些研究为生物分子的细胞内递送提供了新的工具，并有助于设计更有效的细胞穿透蛋白。
We describe a new cell-penetrating protein, B1, capable of delivering conjugated proteins and nucleic acids into mammalian cells. B1 is a 244-amino-acid product of a single-base frameshift in the gene encoding enhanced green fluorescent protein (eGFP). The molecule has a net positive charge of 43 and a very high charge-to-mass ratio of 1.5. eGFP-fused B1 potently penetrates both adherent and suspension cells with >80% of cells taking up the protein when exposed to concentrations as low as 1 μM. The protein was found to cluster in the paranuclear region of TZM-bl cells. Most importantly, we show that B1 not only facilitates cellular uptake, but allows biomolecular cargo to reach sites of biological relevance. For example, baby hamster kidney cells underwent DNA recombination when exposed to B1-tagged Cre recombinase at protein concentrations as low as 2.5 μM, indicating potent nuclear delivery of functional protein cargos. Additionally, B1 delivers non-covalently conjugated RNA and DNA across the cell membrane to cytosolic and nuclear sites accessible to the cellular translation and transcription machinery, as gauged by detection of encoded reporter functions, with efficiency comparable to commercially available cationic lipid reagents. B1 appears to utilize cell-surface glycans and multiple competing endocytic pathways to enter and traffic through cells. These studies provide both a new tool for intracellular delivery of biomolecules and insights that could aid in the design of more effective cell penetrating proteins.
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