Visone Valeria, Han Wenyuan, Perugino Giuseppe, Del Monaco Giovanni, She Qunxin, Rossi Mosè, Valenti Anna, Ciaramella Maria
Institute of Biosciences and Bioresources, National Research Council of Italy, Napoli, Italy.
Department of Biology, University of Copenhagen, Copenhagen, Denmark.
PLoS One. 2017 Oct 3;12(10):e0185791. doi: 10.1371/journal.pone.0185791. eCollection 2017.
Protein imaging, allowing a wide variety of biological studies both in vitro and in vivo, is of great importance in modern biology. Protein and peptide tags fused to proteins of interest provide the opportunity to elucidate protein location and functions, detect protein-protein interactions, and measure protein activity and kinetics in living cells. Whereas several tags are suitable for protein imaging in mesophilic organisms, the application of this approach to microorganisms living at high temperature has lagged behind. Archaea provide an excellent and unique model for understanding basic cell biology mechanisms. Here, we present the development of a toolkit for protein imaging in the hyperthermophilic archaeon Sulfolobus islandicus. The system relies on a thermostable protein tag (H5) constructed by engineering the alkylguanine-DNA-alkyl-transferase protein of Sulfolobus solfataricus, which can be covalently labeled using a wide range of small molecules. As a suitable host, we constructed, by CRISPR-based genome-editing technology, a S. islandicus mutant strain deleted for the alkylguanine-DNA-alkyl-transferase gene (Δogt). Introduction of a plasmid-borne H5 gene in this strain led to production of a functional H5 protein, which was successfully labeled with appropriate fluorescent molecules and visualized in cell extracts as well as in Δogt live cells. H5 was fused to reverse gyrase, a peculiar thermophile-specific DNA topoisomerase endowed with positive supercoiling activity, and allowed visualization of the enzyme in living cells. To the best of our knowledge, this is the first report of in vivo imaging of any protein of a thermophilic archaeon, filling an important gap in available tools for cell biology studies in these organisms.
蛋白质成像能够在体外和体内进行各种各样的生物学研究，在现代生物学中具有重要意义。与感兴趣的蛋白质融合的蛋白质和肽标签为阐明蛋白质的定位和功能、检测蛋白质-蛋白质相互作用以及测量活细胞中的蛋白质活性和动力学提供了机会。虽然有几种标签适用于嗜温生物中的蛋白质成像，但这种方法在高温微生物中的应用却滞后了。古菌为理解基本细胞生物学机制提供了一个优秀且独特的模型。在此，我们展示了一种用于嗜热古菌冰岛硫化叶菌蛋白质成像的工具包的开发。该系统依赖于一种热稳定的蛋白质标签（H5），它是通过对嗜热栖热硫化叶菌的烷基鸟嘌呤-DNA-烷基转移酶蛋白进行工程改造构建而成的，这种标签可以使用多种小分子进行共价标记。作为合适的宿主，我们通过基于CRISPR的基因组编辑技术构建了一种缺失烷基鸟嘌呤-DNA-烷基转移酶基因（Δogt）的冰岛硫化叶菌突变株。在该菌株中引入携带H5基因的质粒导致产生了一种功能性H5蛋白，该蛋白成功地用合适的荧光分子进行了标记，并在细胞提取物以及Δogt活细胞中得以可视化。H5与反向回旋酶融合，反向回旋酶是一种特殊的嗜热菌特异性DNA拓扑异构酶，具有正超螺旋活性，并且能够在活细胞中对该酶进行可视化。据我们所知，这是嗜热古菌任何一种蛋白质体内成像的首次报道，填补了这些生物体细胞生物学研究现有工具中的一个重要空白。
