The method for purifying actin from maize pollen by poly-L-proline-sepharose affinity chromatography was modified by improving some of the experimental conditions (concentration of ATP and K+) and eliminating the use of recombinant human platelet profilin. SDS-PAGE, Western blotting, electron microscopy and ultraviolet absorption analysis demonstrated that the quality of the purified actin was similar to that obtained with the previous method. The yield was 73.5% of that of the previous method. However, the time required for preparation was 2/3 of that in the previous method and the cost of purifying recombinant profilin was saved. Furthermore, a large amount of purified pollen profilin could be obtained at the same time.
ATP和钾离子对肌动蛋白与前纤维蛋白结合的影响及其在植物肌动蛋白纯化中的应用
易克喜1 ! 陶志华1 任海云1,2
（1. 北京师范大学生命科学学院细胞增殖及调控生物学教育部重点实验室，北京100875；
2. 中国农业大学生物学院植物生理生化农业部重点实验室，北京1000094）
摘要：根据ATP和钾离子影响肌动蛋白聚合及肌动蛋白与前纤维蛋白结合的实验结果,通过对以往报道的利用多聚脯氨酸_琼脂糖亲和柱层析法纯化植物肌动蛋白过程中ATP及钾离子浓度的改变 ,不需加外源前纤维蛋白,可得到较大量高纯度具活性的花粉肌动蛋白。SDS凝胶电泳、免疫印迹、电镜负染及紫外检测表明,所得肌动蛋白在纯度、聚合特性等方面均与原方法所得结果相同。肌动蛋白产率为原方法的73.5%,而整个肌动蛋白提纯过程所需时间缩短了1/3, 节省了纯化重组前纤维蛋白所需的费用,消除了外源前纤维蛋白的使用对一些实验室的限制,而且同时得到了大量纯化的花粉前纤维蛋白。
关键词： 肌动蛋白；前纤维蛋白；玉米花粉；多聚脯氨酸-琼脂糖柱层析
全 文 ：