Sustarsic Marko, Plochowietz Anne, Aigrain Louise, Yuzenkova Yulia, Zenkin Nikolay, Kapanidis Achillefs
Clarendon Laboratory, Biological Physics Research Group, Department of Physics, University of Oxford, Parks Road, Oxford, OX1 3PU, UK,
Histochem Cell Biol. 2014 Jul;142(1):113-24. doi: 10.1007/s00418-014-1213-2. Epub 2014 Apr 3.
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 live E. 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.
研究活细胞中蛋白质的结构和动力学对于理解其生理活性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使此类体内研究成为可能；然而，仍存在一些技术挑战。最近，我们开发了一种基于电穿孔的内化方案，该方案可使标记有有机荧光团的生物分子高效导入活的大肠杆菌中（Crawford等人，《生物物理杂志》，2013年，第105卷，第11期，第2439 - 2450页）。在此，我们解决与蛋白质内化相关的重要挑战，并在以下方面优化我们的方法：（1）电穿孔缓冲液条件；（2）从蛋白质储备样品中去除染料污染物；（3）电穿孔后从细胞悬液中去除未内化的分子。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。只要进行了建议的对照实验，使用我们的方法可以内化任何大小达60 kDa的荧光标记蛋白质。此外，我们探究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升高而增加，但细胞活力会受到损害。然而，由于我们样品中受损细胞数量较少，加载细胞的主要部分始终对应于未受损细胞。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Förster共振能量转移对蛋白质扩散、定位和分子内动力学进行体内测量。
