Department of Genetics, University of Osnabrück, Barbarastrasse 11, 49076 Osnabrück, Germany.
Yeast. 2011 Jul;28(7):535-45. doi: 10.1002/yea.1856. Epub 2011 May 18.
The investigation of protein-protein interactions is an essential part of biological research. To obtain a deeper insight into regulatory protein networks, the identification of the components, domains and especially single residues that are involved in these interactions is helpful. A widespread and attractive genetic tool for investigation of protein-protein interactions is the yeast two-hybrid system. This method enables large-scale screens and its application is cheap and relatively simple. For identification of the amino acids in a protein sequence that are essential for interaction with a specific partner, yeast two-hybrid assays can be combined with random mutagenesis of the sequence of interest. A common problem with such an experiment is the generation of stop codons within the mutagenized fragments, leading to the isolation of many false positives when screening for loss of interaction using the two-hybrid method. To overcome this problem, we modified the yeast two-hybrid system to allow selection for sequences without stop codons. To achieve this, we fused the ScURA3 marker-gene in frame to the mutagenized fragments. We show here that this marker is fully functional when fused to a two-hybrid construct with a nuclear localization signal, such as a Gal4 activation domain and a prey protein, thus allowing selection of stop-free sequences on media without uracil. Using the Rho-binding domain from a Bni1-like formin and different Rho-type GTPases from Ashbya gossypii as examples, we further show that our system can be used to screen large numbers of transformants for loss of protein-protein interactions in combination with random mutagenesis.
蛋白质-蛋白质相互作用的研究是生物研究的重要组成部分。为了更深入地了解调控蛋白网络，确定参与这些相互作用的组件、结构域和特别是单个残基是有帮助的。酵母双杂交系统是研究蛋白质-蛋白质相互作用的一种广泛而有吸引力的遗传工具。这种方法可以进行大规模筛选，应用成本低，相对简单。为了确定蛋白质序列中与特定伴侣相互作用所必需的氨基酸，可以将酵母双杂交测定与感兴趣的序列的随机诱变结合使用。这种实验的一个常见问题是在诱变片段中产生终止密码子，当使用双杂交方法筛选相互作用丧失时，会导致产生许多假阳性。为了解决这个问题，我们修改了酵母双杂交系统，以允许选择没有终止密码子的序列。为此，我们将 ScURA3 标记基因与诱变片段融合在一起。我们在这里表明，当与具有核定位信号（如 Gal4 激活结构域和诱饵蛋白）的双杂交构建体融合时，该标记完全有功能，从而可以在不含尿嘧啶的培养基上选择无终止序列。我们以 Bni1 样形成蛋白的 Rho 结合结构域和来自 Ashbya gossypii 的不同 Rho 型 GTPase 为例，进一步表明我们的系统可用于与随机诱变结合筛选大量转化子中蛋白质-蛋白质相互作用的丧失。
