Zhong Jinhui, Zhang Huamei, Stanyon Clement A, Tromp Gerard, Finley Russell L
Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Genome Res. 2003 Dec;13(12):2691-9. doi: 10.1101/gr.1134603. Epub 2003 Nov 12.
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.
描绘蛋白质之间二元相互作用的图谱已成为理解蛋白质如何协同作用以介导生物过程的重要工具。检测具有生物学重要性的蛋白质相互作用的最有效方法之一是酵母双杂交系统。在此，我们提出一种高效的双杂交策略，以促进在全基因组范围内构建蛋白质相互作用图谱。该策略始于两组酵母阵列，其中一组酵母表达与DNA结合结构域（BD）融合的已知蛋白质，另一组酵母表达与转录激活结构域（AD）融合的已知蛋白质。使用可调控的启动子条件性表达融合蛋白，这些启动子在酵母阵列构建和扩增过程中可被抑制。相互作用检测分两个阶段进行。在第一阶段，将少量AD菌株池与BD菌株阵列进行交配。在第二阶段，将单个BD菌株与AD阵列中与第一阶段阳性池相对应的适当子集进行交配。与先前描述的方法相比，该策略具有几个优点，包括能够检测与作为BD融合蛋白抑制酵母生长或激活转录的蛋白质之间的相互作用。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。我们还提出了一种三维池化方案，以进一步提高大规模双杂交分析的效率。
