A strategy for constructing large protein interaction maps using the yeast two-hybrid system: regulated expression arrays and two-phase mating.
A strategy for constructing large protein interaction maps using the yeast two-hybrid system: regulated expression arrays and two-phase mating.
复制标题
使用酵母双杂交系统构建大型蛋白质相互作用图谱的策略：调节表达阵列和两相交配。
DOI：
10.1101/gr.1134603
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发表时间：
2003
期刊：
影响因子：
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通讯作者：
FinleyJr,RussellL
中科院分区：
文献类型：
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作者：
Zhong,Jinhui;Zhang,Huamei;Stanyon,ClementA;Tromp,Gerard;FinleyJr,RussellL
代表蛋白质间二元相互作用的图谱已成为理解蛋白质如何协同工作以介导生物过程的宝贵工具。酵母双杂交系统是检测生物学上重要的蛋白质相互作用的最有效的方法之一。在这里，我们提出了一个有效的双杂交策略，以促进构建蛋白质相互作用的全基因组范围内的地图。该策略开始于两个酵母阵列，其表达与DNA结合结构域（BD）或转录激活结构域（AD）融合的已知蛋白质。融合蛋白使用可在酵母阵列的构建和扩增期间被抑制的受调控启动子进行条件性表达。相互作用试验分两个阶段进行。在第一阶段，AD菌株的小池与BD菌株的阵列交配。在第二阶段中，将单个BD菌株与AD阵列的适当子集配对，所述AD阵列对应于第一阶段中的阳性池。该策略与先前描述的方法相比具有几个优点，包括检测与抑制酵母生长或激活BD融合物转录的蛋白质的相互作用的能力。此外，通过最小化测试大量二元相互作用所需的交配操作和测序反应的数量，该策略比矩阵或文库筛选方法更有效。我们还提出了一个三维池计划，以进一步提高大规模的双杂交分析的效率。
Maps representing the binary interactions among proteins have become valuable tools for understanding how proteins work together to mediate biological processes. One of the most effective methods for detecting biologically important protein interactions has been the yeast two-hybrid system. Here we present an efficient two-hybrid strategy to facilitate construction of protein interaction maps on a genome-wide scale. The strategy begins with two arrays of yeast expressing known proteins fused to either a DNA binding domain (BD), or a transcription activation domain (AD). The fusion proteins are conditionally expressed using regulated promoters that can be repressed during construction and amplification of the yeast arrays. Interaction assays are conducted in two phases. In the first phase, small pools of AD strains are mated with the array of BD strains. In the second phase, individual BD strains are mated with appropriate subsets of the AD array corresponding to positive pools in the first phase. This strategy has several advantages over previously described approaches, including the ability to detect interactions with proteins that inhibit yeast growth or that activate transcription as BD fusions. Moreover, by minimizing the number of mating operations and sequencing reactions needed to test large sets of binary interactions, this strategy is more efficient than either matrix or library screening approaches. We also present a three-dimensional pooling scheme to further increase the efficiency of large-scale two-hybrid analyses.