Increasing specificity in high-throughput yeast two-hybrid experiments
Increasing specificity in high-throughput yeast two-hybrid experiments
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DOI：
10.1016/j.ymeth.2003.10.001
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发表时间：
2004-04-01
期刊：
影响因子：
4.8
通讯作者：
Vidal, M
中科院分区：
文献类型：
--
作者：
Vidalain, PO;Boxem, M;Vidal, M
酵母双杂交（Y2 H）系统自问世以来，已被证明是鉴定新型蛋白质-蛋白质相互作用的有效系统。然而，Y2 H筛查有时因产生高假阳性率而受到批评。最小化假阳性相互作用在蛋白质组宽高通量（HT）Y2 H中尤其重要。在这里，我们总结了在HT-Y2 H项目中减少误报的各种方法。我们评估了通过阴性选择去除猎物编码质粒后检查推定阳性的潜力。我们发现，这种方法可靠地识别由诱饵自发转化为自动激活剂引起的假阳性，并在HT筛选中节省了大量时间。此外，我们提出了一种方法来消除假阳性的一个重要来源：污染的猎物质粒。由于在单个酵母菌落的细胞中存在两种或更多种不同的质粒，Y2 H相互作用物可能被错误地鉴定。在这些独立的质粒中，只有一个编码真正的相互作用因子。通过在相互作用的阳性选择下延长酵母细胞的培养来消除污染质粒，从而允许鉴定真正的相互作用伴侣。(C)2003年爱思唯尔公司All rights reserved.
Since its inception, the yeast two-hybrid (Y2H) system has proven to be an efficient system to identify novel protein-protein interactions. However, Y2H screens are sometimes criticized for generating high rates of false-positives. Minimizing false-positive interactions is especially important in proteome wide high-throughput (HT) Y2H. Here, we Summarize various approaches that reduce false-positives in HT-Y2H projects. We evaluated the potential of examining putative positives after removing the prey encoding plasmid by negative selection. We found that this method reliably identifies false-positives caused by spontaneous conversion of baits into auto-activators and provides significant time-savings in HT screens. In addition, we present a method to eliminate an important source of false-positives: contaminating prey plasmids. Y2H interactors can be wrongly identified due to the presence of two or more different plasmids in the cells of a single yeast colony. Of these independent plasmids, only one encodes a genuine interactor. Contaminating plasmids are eliminated by extended culture of yeast cells under positive selection for the interaction, allowing the identification of the true interaction partner. (C) 2003 Elsevier Inc. All rights reserved.