Lipinszki Zoltan, Wang Peng, Grant Rhys, Lindon Catherine, Dzhindzhev Nikola S, D'Avino Pier Paolo, Przewloka Marcin R, Glover David M, Archambault Vincent
Department of Genetics, University of Cambridge, Cambridge, UK.
Methods Mol Biol. 2014;1170:571-88. doi: 10.1007/978-1-4939-0888-2_33.
The ability to identify protein interactions is key to elucidating the molecular mechanisms of cellular processes, including mitosis and cell cycle regulation. Drosophila melanogaster, as a model system, provides powerful tools to study cell division using genetics, microscopy, and RNAi. Drosophila early embryos are highly enriched in mitotic protein complexes as their nuclei undergo 13 rounds of rapid, synchronous mitotic nuclear divisions in a syncytium during the first 2 h of development. Here, we describe simple methods for the affinity purification of protein complexes from transgenic fly embryos via protein A- and green fluorescent protein-tags fused to bait proteins of interest. This in vivo proteomics approach has allowed the identification of several known and novel mitotic protein interactions using mass spectrometry, and it expands the use of the Drosophila model in modern molecular biology.
识别蛋白质相互作用的能力是阐明细胞过程分子机制的关键，这些细胞过程包括有丝分裂和细胞周期调控。黑腹果蝇作为一种模式系统，提供了利用遗传学、显微镜技术和RNA干扰来研究细胞分裂的强大工具。果蝇早期胚胎富含有丝分裂蛋白复合物，因为在发育的最初2小时内，其细胞核在合胞体中经历13轮快速、同步的有丝分裂核分裂。在此，我们描述了通过与感兴趣的诱饵蛋白融合的蛋白A和绿色荧光蛋白标签，从转基因果蝇胚胎中亲和纯化蛋白复合物的简单方法。这种体内蛋白质组学方法已通过质谱鉴定出几种已知的和新的有丝分裂蛋白相互作用，并且扩展了果蝇模型在现代分子生物学中的应用。
