Letteboer Stef J F, Roepman Ronald
Department of Human Genetics Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
Methods Mol Biol. 2008;484:145-59. doi: 10.1007/978-1-59745-398-1_10.
Identification of binary protein-protein interactions is a crucial step in determining the molecular context and functional pathways of proteins. State-of-the-art proteomics techniques provide high-throughput information on the content of proteomes and protein complexes, but give little information about transient interactions, about the binary protein pairs, or about the interacting epitopes. A powerful method to reveal this information is the yeast two-hybrid system. We have employed an optimized GAL4-based yeast two-hybrid system to dissect the photoreceptor cilium-associated protein complex around the retinitis pigmentosa GTPase regulator (RPGR) in mammalian photoreceptors. This enabled us to identify associating protein partners that, similar to RPGR, were also associated with a heterogeneous group of inherited retinal degenerations arising from ciliary defects. We describe how to generate high content pretransformed cDNA libraries, and perform an efficient yeast mating screen for protein-protein interactions with any bait protein of interest.
鉴定二元蛋白质-蛋白质相互作用是确定蛋白质分子背景和功能途径的关键步骤。最先进的蛋白质组学技术可提供有关蛋白质组和蛋白质复合物含量的高通量信息，但关于瞬时相互作用、二元蛋白质对或相互作用表位的信息却很少。揭示此类信息的一种强大方法是酵母双杂交系统。我们采用了一种基于GAL4的优化酵母双杂交系统，剖析哺乳动物光感受器中视网膜色素变性GTP酶调节蛋白（RPGR）周围的光感受器纤毛相关蛋白复合物。这使我们能够鉴定出与RPGR类似的、也与因纤毛缺陷引起的一组异质性遗传性视网膜变性相关的缔合蛋白质伴侣。我们描述了如何生成高含量的预转化cDNA文库，以及如何针对与任何感兴趣的诱饵蛋白的蛋白质-蛋白质相互作用进行高效的酵母交配筛选。
