Department of Life Sciences, Ben-Gurion University of the Negev, Beer Sheva, Israel.
National Institute for Biotechnology in the Negev (NIBN), Ben-Gurion University of the Negev, Beer Sheva, Israel.
FEBS J. 2021 Feb;288(4):1107-1117. doi: 10.1111/febs.15477. Epub 2020 Jul 22.
Twenty-five years ago, GFP revolutionized the field of cell biology by enabling scientists to visualize, for the first time, proteins in living cells. However, when it comes to current, state-of-the-art imaging technologies, fluorescent proteins (such as GFP) have several limitations that result from their size and photophysics. Over the past decade, an elegant, alternative approach, which is based on the direct labeling of proteins with fluorescent dyes and is compatible with live-cell and super-resolution imaging applications, has been introduced. In this approach, an unnatural amino acid that can covalently bind a fluorescent dye is incorporated into the coding sequence of a protein. The protein of interest is thereby site-specifically fluorescently labeled inside the cell, eliminating the need for protein- or peptide-labeling tags. Whether this labeling approach will change cell biology research is currently unclear, but it clearly has the potential to do so. In this short review, a general overview of this approach is provided, focusing on the imaging of site-specifically labeled proteins in mammalian tissue culture cells, and highlighting its advantages and limitations for cellular imaging.
25 年前，GFP 革命性地改变了细胞生物学领域，使科学家们能够首次在活细胞中可视化蛋白质。然而，就当前最先进的成像技术而言，荧光蛋白（如 GFP）因其大小和光物理性质而存在一些局限性。在过去的十年中，一种优雅的替代方法被引入，该方法基于用荧光染料直接标记蛋白质，并且与活细胞和超分辨率成像应用兼容。在这种方法中，将可以共价结合荧光染料的非天然氨基酸掺入到蛋白质的编码序列中。因此，感兴趣的蛋白质在细胞内被特异性荧光标记，无需使用蛋白质或肽标记标签。这种标记方法是否会改变细胞生物学研究目前尚不清楚，但它显然有潜力做到这一点。在这篇简短的综述中，提供了该方法的概述，重点介绍了在哺乳动物组织培养细胞中特异性标记蛋白质的成像，并强调了其用于细胞成像的优势和局限性。
