An efficient screening method for purifying and crystallizing membrane proteins using modified clear-native PAGE
An efficient screening method for purifying and crystallizing membrane proteins using modified clear-native PAGE
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使用改良的透明天然 PAGE 纯化和结晶膜蛋白的有效筛选方法
DOI：
10.1016/j.ab.2018.02.007
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发表时间：
2018
影响因子：
2.9
通讯作者：
Murata Takeshi
中科院分区：
文献类型：
--
作者：
Suzuki Nanao;Takamuku Yuuki;Asakawa Tomohiro;Inai Makoto;Hino Tomoya;Iwata So;Kan Toshiyuki;Murata Takeshi
膜蛋白，如G蛋白偶联受体，控制细胞与其环境之间的通讯，是许多细胞功能不可或缺的。然而，对膜蛋白的结构研究落后于对水溶性蛋白的研究，这是由于它们的结构稳定性低，使得难以获得用于X射线晶体学的晶体。优化条件以提高膜蛋白的稳定性是成功结晶的关键。然而，优化通常需要大量纯化的样品，并且这是一个耗时且反复试验的过程。在这里，我们报告了一种快速的方法，使用透明的天然聚丙烯酰胺凝胶电泳（CN-PAGE）与修改后的考马斯亮蓝G-250（mCBB）染色，在甲酸钠中减少膜蛋白的预沉淀筛选。选择A2 Aadenosine receptor（A2 AAR）作为靶膜蛋白，我们先前使用抗体获得了其晶体结构，并表达为用于凝胶内荧光检测的红色荧光蛋白融合体。mCBB CN-PAGE方法能够使用表达蛋白质的增溶膜级分优化A2 AAR的增溶、纯化和结晶条件，而无需纯化程序。这些数据表明，mCBB CN-PAGE技术的适用性，以各种各样的整合膜蛋白。
Membrane proteins, such as G-protein coupled receptors, control communication between cells and their environments and are indispensable for many cellular functions. Nevertheless, structural studies on membrane proteins lag behind those on water-soluble proteins, due to their low structural stability, making it difficult to obtain crystals for X-ray crystallography. Optimizing conditions to improve the stability of membrane proteins is essential for successful crystallization. However, the optimization usually requires large amounts of purified samples, and it is a time-consuming and trial-and-error process. Here, we report a rapid method for precrystallization screening of membrane proteins using Clear Native polyacrylamide gel electrophoresis (CN-PAGE) with the modified Coomassie Brilliant Blue G-250 (mCBB) stain that was reduced in sodium formate. A2Aadenosine receptor (A2AAR) was selected as a target membrane protein, for which we previously obtained the crystal structure using an antibody, and was expressed as a red fluorescent protein fusion for in-gel fluorescence detection. The mCBB CN-PAGE method enabled the optimization of the solubilization, purification, and crystallization conditions of A2AAR using the solubilized membrane fraction expressing the protein without purification procedures. These data suggest the applicability of mCBB CN-PAGE technique to a wide variety of integral membrane proteins.