Department of Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
J Proteome Res. 2013 Jul 5;12(7):3181-92. doi: 10.1021/pr400047p. Epub 2013 Jun 17.
Physical interactions between proteins are essential for biological processes. Hence, there have been major efforts to elucidate the complete networks of protein-protein interactions, or "interactomes", of various organisms. Detailed descriptions of protein interaction networks should include information on the discrete domains that mediate these interactions, yet most large-scale efforts model interactions between whole proteins only. We previously developed a yeast two-hybrid-based strategy to systematically map interaction domains and generated a domain-based interactome network for 750 proteins involved in C. elegans early embryonic development. Here, we expand the concept of Y2H-based interaction domain mapping to the genome-wide level. We generated a human fragment library by randomly fragmenting the full-length open reading frames (ORFs) present in the human ORFeome collection. Screens using several proteins required for cell division or polarity establishment as baits demonstrate the ability to accurately identify interaction domains for human proteins using this approach, while the experimental quality of the Y2H data was independently verified in coaffinity purification assays. The library generation strategy can easily be adapted to generate libraries from full-length ORF collections of other organisms.
蛋白质之间的物理相互作用对于生物过程至关重要。因此，人们已经做出了重大努力来阐明各种生物体的完整蛋白质-蛋白质相互作用网络，或“相互作用组”。蛋白质相互作用网络的详细描述应该包括介导这些相互作用的离散结构域的信息，但大多数大规模的研究仅对整个蛋白质之间的相互作用进行建模。我们之前开发了一种基于酵母双杂交的策略，系统地绘制了相互作用结构域图谱，并为参与秀丽隐杆线虫早期胚胎发育的 750 种蛋白质生成了基于结构域的相互作用组网络。在这里，我们将基于 Y2H 的相互作用结构域映射的概念扩展到全基因组水平。我们通过随机片段化人类 ORFeome 集合中存在的全长开放阅读框 (ORF) 生成了一个人类片段文库。使用几个细胞分裂或极性建立所需的蛋白质作为诱饵进行的筛选证明了使用这种方法准确识别人类蛋白质相互作用结构域的能力，而 Y2H 数据的实验质量在共亲和纯化测定中独立得到了验证。文库生成策略可以轻松地适应于从其他生物体的全长 ORF 集合中生成文库。
