Lievens Sam, Vanderroost Nele, Van der Heyden José, Gesellchen Viola, Vidal Marc, Tavernier Jan
Department of Medical Protein Research, VIB, A. Baertsoenkaai 3, 9000 Ghent, Belgium.
J Proteome Res. 2009 Feb;8(2):877-86. doi: 10.1021/pr8005167.
Physical interactions between proteins play a key role in probably every cellular process. Efforts to chart the protein interaction networks are ongoing in a number of model organisms using a diversity of approaches. The resulting genome-wide interaction maps will provide a scaffold for further detailed functional analysis. We developed MAPPIT, a mammalian two-hybrid approach that allows identification and analysis of mammalian protein-protein interactions in their native environment. Here, we introduce an efficient MAPPIT assay that permits high-throughput screening of arrayed collections of proteins and complements a previously published cDNA library screening approach. We validated both methods in screens for interaction partners of the Cullin-based E3 ubiquitin ligase subunits SKP1 and Elongin C. In addition to a number of known interactors, novel SKP1 and Elongin C binding proteins were identified. The array assay is an important addition to the MAPPIT suite of technologies that is expected to significantly increase its utility as a toolbox to screen for novel interactors of proteins or small molecules.
蛋白质之间的物理相互作用可能在每个细胞过程中都起着关键作用。目前正在使用多种方法，在一些模式生物中绘制蛋白质相互作用网络。由此产生的全基因组相互作用图谱将为进一步的详细功能分析提供一个框架。我们开发了MAPPIT，一种哺乳动物双杂交方法，可用于在天然环境中鉴定和分析哺乳动物蛋白质-蛋白质相互作用。在此，我们介绍一种高效的MAPPIT检测方法，该方法允许对排列的蛋白质文库进行高通量筛选，并补充了先前发表的cDNA文库筛选方法。我们在筛选基于Cullin的E3泛素连接酶亚基SKP1和Elongin C的相互作用伙伴时，对这两种方法进行了验证。除了一些已知的相互作用蛋白外，还鉴定出了新的SKP1和Elongin C结合蛋白。阵列检测是MAPPIT技术套件的一项重要补充，有望显著提高其作为筛选蛋白质或小分子新相互作用蛋白的工具箱的效用。
