One-step purification of assembly-competent tubulin from diverse eukaryotic sources.
One-step purification of assembly-competent tubulin from diverse eukaryotic sources.
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DOI：
10.1091/mbc.e12-06-0444
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发表时间：
2012-11
影响因子：
3.3
通讯作者：
Drechsel DN
中科院分区：
文献类型：
--
作者：
Widlund PO;Podolski M;Reber S;Alper J;Storch M;Hyman AA;Howard J;Drechsel DN
提出了一种方法，允许快速和有效的纯化，活性微管蛋白从各种物种和组织来源亲和层析。它消除了使用异源系统研究微管相关蛋白和运动蛋白的需要，这一直是微管相关研究的主要问题。我们已经开发了一种协议，允许通过亲和层析法从各种物种和组织来源中快速有效地纯化天然活性微管蛋白。亲和基质包括细菌表达的重组蛋白，即来自酿酒酵母Stu2的TOG1/2结构域，共价偶联到Sepharose载体上。该树脂具有从澄清的粗细胞提取物中特异性结合微管蛋白的高能力，并且在洗涤后，高纯度的微管蛋白可以在温和的条件下洗脱。洗脱后的微管蛋白功能齐全，可以有效地组装成微管。该方法消除了使用异源系统研究微管相关蛋白和运动蛋白的需要，这一直是微管相关研究的主要问题。
A method is presented that allows rapid and efficient purification of native, active tubulin from a variety of species and tissue sources by affinity chromatography. It eliminates the need to use heterologous systems for the study of microtubule-associated proteins and motor proteins, which has been a major issue in microtubule-related research. We have developed a protocol that allows rapid and efficient purification of native, active tubulin from a variety of species and tissue sources by affinity chromatography. The affinity matrix comprises a bacterially expressed, recombinant protein, the TOG1/2 domains from Saccharomyces cerevisiae Stu2, covalently coupled to a Sepharose support. The resin has a high capacity to specifically bind tubulin from clarified crude cell extracts, and, after washing, highly purified tubulin can be eluted under mild conditions. The eluted tubulin is fully functional and can be efficiently assembled into microtubules. The method eliminates the need to use heterologous systems for the study of microtubule-associated proteins and motor proteins, which has been a major issue in microtubule-related research.