Vemu Annapurna, Garnham Christopher P, Lee Duck-Yeon, Roll-Mecak Antonina
Cell Biology and Biophysics Unit, National Institute of Neurological Disorders and Stroke, Bethesda, Maryland, USA.
Biochemistry Core, National Heart, Lung and Blood Institute, Bethesda, Maryland, USA.
Methods Enzymol. 2014;540:149-66. doi: 10.1016/B978-0-12-397924-7.00009-1.
Tubulin, the building block of microtubules, is subject to chemically diverse and evolutionarily conserved post-translational modifications that mark microtubules for specific functions in the cell. Here we describe in vitro methods for generating homogenous acetylated, glutamylated, or tyrosinated tubulin and microtubules using recombinantly expressed and purified modification enzymes. The generation of differentially modified microtubules now enables a mechanistic dissection of the effects of tubulin post-translational modifications on the dynamics and mechanical properties of microtubules as well as the behavior of motors and microtubule-associated proteins.
微管蛋白是微管的组成成分，会发生化学性质多样且在进化上保守的翻译后修饰，这些修饰为微管在细胞中的特定功能打上标记。在此，我们描述了利用重组表达和纯化的修饰酶体外生成均匀乙酰化、谷氨酰化或酪氨酸化微管蛋白及微管的方法。现在，生成差异修饰的微管能够对微管蛋白翻译后修饰对微管动力学和力学性质以及马达蛋白和微管相关蛋白行为的影响进行机制剖析。
