Hotta Takashi, Hashimoto Takashi
Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, United States.
Division of Biological Science, Nara Institute of Science and Technology, Ikoma, Nara, Japan.
Methods Cell Biol. 2020;160:263-280. doi: 10.1016/bs.mcb.2020.06.002. Epub 2020 Jul 22.
In the plant cytoskeleton research, mammalian brain tubulin has been widely used to study plant microtubule-interacting proteins in vitro since purification of tubulins from plant sources is generally considered to be challenging and time-consuming. A convenient method for affinity purification of tubulins was devised, which utilized the TOG domains of yeast Stu2 tubulin-binding protein as an affinity ligand (Widlund et al., 2012). We showed that this so-called TOG tubulin affinity chromatography worked efficiently with plant materials, especially actively-dividing cultured cells (Hotta et al., 2016). Plant tubulins purified with the TOG method is highly assembly-competent and thus can be used in various in vitro experiments. Here, we summarize purification strategies of native or tagged plant tubulins as well as an in vitro pull-down assay to monitor their polymerization activity.
在植物细胞骨架研究中，由于从植物来源纯化微管蛋白通常被认为具有挑战性且耗时，哺乳动物脑微管蛋白已被广泛用于体外研究植物微管相互作用蛋白。人们设计了一种便捷的微管蛋白亲和纯化方法，该方法利用酵母Stu2微管蛋白结合蛋白的TOG结构域作为亲和配体（维德伦德等人，2012年）。我们表明，这种所谓的TOG微管蛋白亲和色谱法对植物材料有效，尤其是对活跃分裂的培养细胞（堀田等人，2016年）。用TOG方法纯化的植物微管蛋白具有高度组装能力，因此可用于各种体外实验。在此，我们总结了天然或标记植物微管蛋白的纯化策略以及用于监测其聚合活性的体外下拉分析方法。
