优化的Native PAGE蛋白回收方法在AFM中的应用

Application of Optimized Method of Protein Extraction from Native PAGE for Atomic Force Microscopy

原子力显微镜(AFM)可以在生理条件下获得生物大分子的自然结构信息而不需要进行图像处理。虽然对含有大小范围不一的异质样品成像可以得到几纳米的分辨率,但是具有更高的亚纳米级分辨率的图像仅可以从含有单一亚基数目的高度均一的蛋白复合物获得。在重组蛋白的制备中这种蛋白复合物经常不存在,尤其是对于膜蛋白则更具有挑战性。本研究中,研究者以一种熟知的膜成孔蛋白复合物Perfringolysin O(PFO)作为模型系统,研制出一种方法来分离含有单一亚基数目的复合物,并用于随后在液相的AFM成像。具体而言,研究人员通过非变性聚丙烯酰胺凝胶电泳(Native PAGE)分离出该复合物中特定大小的蛋白成分,然后提取时采用一种新方法使结构完整保持。预计这种方法不仅对更多其他蛋白的高分辨率AFM研究有效用,而且在基于单分子方法和生物技术应用的研究上有更普遍的用途。

Atomic force microscopy(AFM)can determine structural information of biological macromolecules under physiological conditions directly from unprocessed images.However,while images with a resolution of a few nanometers can be obtained from heterogeneous samples containing macromolecules with a range of sizes and stoichiometries,higher resolution images with sub-nanometer-scale details have only been obtained from highly homogeneous protein complexes of a single stoichiometry,which is frequently not present in preparations of recombinant proteins and is especially challenging with membrane proteins.Here,using perfringolysin O(PFO),a well-studied membrane pore-forming protein that is well known to form a wide range of stoichiometries,as a model system,we had developed a strategy to isolate a single-sized complex that was suitable for subsequent AFM imaging under solution.In particular,we isolated a specific size of this complex using native polyacrylamide gel electrophoresis(Native PAGE),followed by a structure-preserving method to extract the isolated complex.We anticipated that this method would prove useful not only for high resolution AFM studies of many other proteins,but also,more generally,for studies using other single-molecule based methods and biotechnological applications.