Increasing specificity in high-throughput yeast two-hybrid experiments
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- 作者：Vidalain ; Pierre-Olivier ; Boxem ; Mike ; Ge ; Hui ; Li ; Siming ; Vidal ; Marc
- 关键词：Yeast ; Two-hybrid ; False-positives ; High-throughput ; Large-scale ; Genomics ; Proteomics
- 刊名：Methods
- 出版年：2004
- 出版时间：April, 2004
- 年：2004
- 卷：32
- 期：4
- 页码：363-370
- 全文大小：377 K
文摘
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.