Optimized delivery of fluorescently labeled proteins in live bacteria using electroporation.
Optimized delivery of fluorescently labeled proteins in live bacteria using electroporation.
复制标题
使用电穿孔优化活细菌中荧光标记蛋白质的递送。
DOI：
10.1007/s00418-014-1213-2
复制
发表时间：
2014
影响因子：
2.3
通讯作者：
Sustarsic M
中科院分区：
文献类型：
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作者：
Sustarsic M
研究活细胞中蛋白质的结构和动力学对于了解其生理活动和机制以及验证体外表征至关重要。标记和成像技术的改进开始允许此类体内研究；然而，仍然存在一些技术挑战。最近，我们开发了一种基于电穿孔的内化方案，该方案允许将有机荧光团标记的生物分子高效引入 liveE 中。大肠杆菌（Crawford 等人，Biophys J 105 (11):2439–2450, 2013）。在这里，我们解决了与蛋白质内化相关的重要挑战，并在（1）电穿孔缓冲液条件方面优化了我们的方法； (2) 去除蛋白质储备样品中的染料污染物； (3)电穿孔后从细胞悬液中去除非内化分子。我们通过展示 10-kDa 蛋白质（RNA 聚合酶的 ω 亚基）的高效内化来说明优化方案的可用性。如果进行了建议的对照实验，任何高达 60 kDa 的荧光标记蛋白质都可以使用我们的方法内化。此外，我们探讨了电穿孔电压对内化效率和细胞活力的影响，并证明，虽然内化随着电压的增加而增加，但细胞活力受到损害。然而，由于我们的样品中受损细胞的数量较少，加载的细胞的主要部分始终对应于未受损的细胞。通过注意仅将活细胞纳入分析，我们的方法允许进行生理相关研究，包括通过单分子福斯特共振能量转移体内测量蛋白质扩散、定位和分子内动力学。
Studying the structure and dynamics of proteins in live cells is essential to understanding their physiological activities and mechanisms, and to validating in vitro characterization. Improvements in labeling and imaging technologies are starting to allow such in vivo studies; however, a number of technical challenges remain. Recently, we developed an electroporation-based protocol for internalization, which allows biomolecules labeled with organic fluorophores to be introduced at high efficiency into liveE. coli(Crawford et al. in Biophys J 105 (11):2439–2450, 2013). Here, we address important challenges related to internalization of proteins, and optimize our method in terms of (1) electroporation buffer conditions; (2) removal of dye contaminants from stock protein samples; and (3) removal of non-internalized molecules from cell suspension after electroporation. We illustrate the usability of the optimized protocol by demonstrating high-efficiency internalization of a 10-kDa protein, the ω subunit of RNA polymerase. Provided that suggested control experiments are carried out, any fluorescently labeled protein of up to 60 kDa could be internalized using our method. Further, we probe the effect of electroporation voltage on internalization efficiency and cell viability and demonstrate that, whilst internalization increases with increased voltage, cell viability is compromised. However, due to the low number of damaged cells in our samples, the major fraction of loaded cells always corresponds to non-damaged cells. By taking care to include only viable cells into analysis, our method allows physiologically relevant studies to be performed, including in vivo measurements of protein diffusion, localization and intramolecular dynamics via single-molecule Förster resonance energy transfer.