Castoldi Mirco, Popov Andrei V
European Molecular Biology Laboratory, Cell Biology and Biophysics Programme, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
Protein Expr Purif. 2003 Nov;32(1):83-8. doi: 10.1016/S1046-5928(03)00218-3.
Microtubules can be assembled in vitro from purified alpha/beta tubulin heterodimers in the presence of GTP. Tubulin is routinely obtained from animal brain tissue through repetitive cycles of polymerization-depolymerization, followed by ion-exchange chromatography to remove any contaminating microtubule-associated proteins and motors. Here, we show that only two cycles of polymerization-depolymerization of pig brain tubulin in the presence of a high-molarity PIPES buffer allow the efficient removal of contaminating proteins and production of a high-concentration tubulin solution. The proposed protocol is rapid and yields more active tubulin than the traditional ion-exchange chromatography-based procedures.
在存在鸟苷三磷酸（GTP）的情况下，微管蛋白可以由纯化的α/β微管蛋白异源二聚体在体外组装而成。微管蛋白通常通过聚合-解聚的重复循环从动物脑组织中获得，随后进行离子交换色谱以去除任何污染的微管相关蛋白和马达蛋白。在此，我们表明，在高摩尔浓度的哌嗪-N,N'-双(2-乙磺酸)（PIPES）缓冲液存在下，猪脑微管蛋白仅经过两个聚合-解聚循环就能有效去除污染蛋白并产生高浓度的微管蛋白溶液。所提出的方案快速，并且比传统的基于离子交换色谱的方法产生更多活性微管蛋白。
