Purification of Tubulin from the Fission Yeast Schizosaccharomyces pombe
Purification of Tubulin from the Fission Yeast Schizosaccharomyces pombe
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DOI：
10.1007/978-1-61779-252-6_3
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发表时间：
2011-01-01
期刊：
影响因子：
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通讯作者：
Cross, Robert A.
中科院分区：
文献类型：
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作者：
Drummond, Douglas R.;Kain, Susan;Cross, Robert A.
裂殖酵母是一种很有吸引力的微管蛋白来源，因为它含有很少的微管蛋白异构体，并且易于进行基因操作。我们描述了从S.pombe中制备高纯度的天然微管蛋白的毫克量，适用于微管动力学分析以及结构和其他生化研究。可溶性蛋白质组分通过批量结合结合到阴离子交换层析树脂上，包装在层析柱中，并用盐梯度洗脱。含有微管蛋白的部分经过硫酸铵沉淀，以进一步浓缩和纯化蛋白质。在聚合和解聚循环之前进行一轮高分辨率阴离子交换层析，以选择功能微管蛋白。凝胶过滤用于在最后的除盐步骤之前去除残留污染物。纯化的微管蛋白被浓缩，然后冷冻并储存在液氮中。
The fission yeast Schizosaccharomyces pombe is an attractive source of tubulin for biochemical experiments as it contains few tubulin isoforms and is amenable to genetic manipulation. We describe the preparation of milligram quantities of highly purified native tubulin from S. pombe suitable for use in microtubule dynamics assays as well as structural and other biochemical studies.S. pombe cells are grown in bulk in a fermenter and then lysed using a bead mill. The soluble protein fraction is bound to anion-exchange chromatography resin by batch binding, packed in a chromatography column and eluted by a salt gradient. The tubulin-containing fraction is ammonium sulphate precipitated to further concentrate and purify the protein. A round of high-resolution anion-exchange chromatography is carried out before a cycle of polymerisation and depolymerisation to select functional tubulin. Gel filtration is used to remove residual contaminants before a final desalting step. The purified tubulin is concentrated, and then frozen and stored in liquid nitrogen.