Bürckstümmer Tilmann, Bennett Keiryn L, Preradovic Adrijana, Schütze Gregor, Hantschel Oliver, Superti-Furga Giulio, Bauch Angela
Research Center for Molecular Medicine (CeMM), Lazarettgasse 19/3, 1090 Vienna, Austria.
Nat Methods. 2006 Dec;3(12):1013-9. doi: 10.1038/nmeth968. Epub 2006 Oct 22.
Tandem affinity purification (TAP) is a generic two-step affinity purification protocol that enables the isolation of protein complexes under close-to-physiological conditions for subsequent analysis by mass spectrometry. Although TAP was instrumental in elucidating the yeast cellular machinery, in mammalian cells the method suffers from a low overall yield. We designed several dual-affinity tags optimized for use in mammalian cells and compared the efficiency of each tag to the conventional TAP tag. A tag based on protein G and the streptavidin-binding peptide (GS-TAP) resulted in a tenfold increase in protein-complex yield and improved the specificity of the procedure. This allows purification of protein complexes that were hitherto not amenable to TAP and use of less starting material, leading to higher success rates and enabling systematic interaction proteomics projects. Using the well-characterized Ku70-Ku80 protein complex as an example, we identified both core elements as well as new candidate effectors.
串联亲和纯化（TAP）是一种通用的两步亲和纯化方案，能够在接近生理条件下分离蛋白质复合物，以便随后通过质谱进行分析。尽管TAP在阐明酵母细胞机制方面发挥了重要作用，但在哺乳动物细胞中，该方法的总体产量较低。我们设计了几种针对哺乳动物细胞使用进行优化的双亲和标签，并将每个标签的效率与传统TAP标签进行了比较。一种基于蛋白G和链霉亲和素结合肽的标签（GS-TAP）使蛋白质复合物产量提高了十倍，并提高了该方法的特异性。这使得以前无法用TAP纯化的蛋白质复合物得以纯化，并且使用更少的起始材料，从而提高成功率并推动系统性相互作用蛋白质组学项目。以特征明确的Ku70-Ku80蛋白复合物为例，我们鉴定出了核心元件以及新的候选效应物。
