Giannone Richard J, McDonald W Hayes, Hurst Gregory B, Huang Ying, Wu Jun, Liu Yie, Wang Yisong
Oak Ridge National Laboratory, Oak Ridge, TN, USA.
Biotechniques. 2007 Sep;43(3):296, 298, 300 passim. doi: 10.2144/000112550.
Although affinity purification coupled with mass spectrometry (MS) provides a powerful tool to study protein-protein interactions, this strategy has encountered numerous difficulties when adapted to mammalian cells. Here we describe a Gateway-compatible dual-tag affinity purification system that integrates regulatable expression, tetracysteine motifs, and various combinations ofaffinity tags to facilitate the cloning, detection, and purification of bait proteins and their interacting partners. Utilizing the human telomere binding protein TRF2 as a benchmark, we demonstrate bait protein recoveries upwards of approximately 16% from as little as 1-7 x 10(7) cells and successfully identify known TRF2 interacting proteins, suggesting that our dual-tag affinity purification approach is a capable new tool for expanding the capacity to explore mammalian proteomic networks.
虽然亲和纯化与质谱联用（MS）为研究蛋白质-蛋白质相互作用提供了强大工具，但该策略应用于哺乳动物细胞时遇到了诸多困难。在此，我们描述了一种与Gateway兼容的双标签亲和纯化系统，该系统整合了可调控表达、四半胱氨酸基序以及多种亲和标签组合，以促进诱饵蛋白及其相互作用伴侣的克隆、检测和纯化。以人类端粒结合蛋白TRF2作为基准，我们证明从低至1 - 7×10⁷个细胞中可回收约16%以上的诱饵蛋白，并成功鉴定出已知的TRF2相互作用蛋白，这表明我们的双标签亲和纯化方法是一种用于扩展探索哺乳动物蛋白质组网络能力的有效新工具。
