Department of Molecular Genetics, University of Toronto, Toronto, Canada.
Methods. 2012 Aug;57(4):409-16. doi: 10.1016/j.ymeth.2012.06.005. Epub 2012 Jun 17.
Protein-protein interactions (PPIs) are central to our understanding of protein function, biological processes and signaling pathways. Affinity purification coupled with mass spectrometry (AP-MS) is a powerful approach for detecting PPIs and protein complexes and relies on the purification of bait proteins using bait-specific binding reagents. These binding reagents may recognize bait proteins directly or affinity tags that are fused to bait proteins. A limitation of the latter approach is that expression of affinity tagged baits is largely constrained to engineered or unnatural cell lines, which results in the AP-MS identification of PPIs that may not accurately reflect those seen in nature. Therefore, generating cell lines stably expressing affinity tagged bait proteins in a broad range of cell types and cell lines is important for identifying PPIs that are dependent on different contexts. To facilitate the identification of PPIs across many mammalian cell types, we developed the mammalian affinity purification and lentiviral expression (MAPLE) system. MAPLE uses recombinant lentiviral technology to stably and efficiently express affinity tagged complementary DNA (cDNA) in mammalian cells, including cells that are difficult to transfect and non-dividing cells. The MAPLE vectors contain a versatile affinity (VA) tag for multi-step protein purification schemes and subcellular localization studies. In this methods article, we present a step-by-step overview of the MAPLE system workflow.
蛋白质-蛋白质相互作用（PPIs）是我们理解蛋白质功能、生物过程和信号通路的核心。亲和纯化结合质谱（AP-MS）是检测蛋白质相互作用和蛋白质复合物的有力方法，它依赖于使用诱饵蛋白特异性结合试剂对诱饵蛋白进行纯化。这些结合试剂可以直接识别诱饵蛋白，也可以识别融合到诱饵蛋白上的亲和标签。后一种方法的一个局限性是，亲和标记诱饵蛋白的表达在很大程度上局限于工程或非自然细胞系，这导致 AP-MS 鉴定的蛋白质相互作用可能不能准确反映自然界中观察到的相互作用。因此，在广泛的细胞类型和细胞系中稳定表达亲和标记诱饵蛋白的细胞系对于鉴定依赖于不同环境的蛋白质相互作用非常重要。为了促进在许多哺乳动物细胞类型中鉴定蛋白质相互作用，我们开发了哺乳动物亲和纯化和慢病毒表达（MAPLE）系统。MAPLE 使用重组慢病毒技术在哺乳动物细胞中稳定而有效地表达亲和标记的 cDNA，包括难以转染和非分裂的细胞。MAPLE 载体包含一个通用的亲和（VA）标签，用于多步蛋白质纯化方案和亚细胞定位研究。在本方法文章中，我们提供了 MAPLE 系统工作流程的分步概述。
