Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
Mol Cell Proteomics. 2012 Aug;11(8):501-11. doi: 10.1074/mcp.O111.016246. Epub 2012 Apr 3.
Determining the localization, binding partners, and secondary modifications of individual proteins is crucial for understanding protein function. Several tags have been constructed for protein localization or purification under either native or denaturing conditions, but few tags permit all three simultaneously. Here, we describe a multifunctional tandem affinity purification (MAP) method that is both highly efficient and enables protein visualization. The MAP tag utilizes affinity tags inserted into an exposed surface loop of mVenus offering two advantages: (1) mVenus fluorescence can be used for protein localization or FACS-based selection of cell lines; and (2) spatial separation of the affinity tags from the protein results in high recovery and reduced variability between proteins. MAP purification was highly efficient in multiple organisms for all proteins tested. As a test case, MAP combined with liquid chromatography-tandem MS identified known and new candidate binding partners and modifications of the kinase Plk1. Thus the MAP tag is a new powerful tool for determining protein modification, localization, and interactions.
确定单个蛋白质的定位、结合伙伴和二级修饰对于理解蛋白质功能至关重要。已经构建了几种标签，用于在天然或变性条件下进行蛋白质定位或纯化，但很少有标签同时允许这三种功能。在这里，我们描述了一种多功能串联亲和纯化 (MAP) 方法，该方法既高效又能够实现蛋白质可视化。MAP 标签利用插入 mVenus 暴露表面环中的亲和标签提供了两个优势：(1) mVenus 荧光可用于蛋白质定位或基于 FACS 的细胞系选择；(2) 亲和标签与蛋白质的空间分离导致高回收率和蛋白质之间的变异性降低。MAP 纯化在多种生物体中对所有测试的蛋白质都非常高效。作为一个测试案例，MAP 与液相色谱-串联质谱结合鉴定了激酶 Plk1 的已知和新候选结合伙伴和修饰。因此，MAP 标签是确定蛋白质修饰、定位和相互作用的新有力工具。
