Reverse two-hybrid techniques in the yeast Saccharomyces cerevisiae.
Reverse two-hybrid techniques in the yeast Saccharomyces cerevisiae.
复制标题
酿酒酵母中的反向二杂交技术。
DOI：
10.1007/978-1-4939-2425-7_28
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Kahn,RichardA
中科院分区：
文献类型：
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作者：
Bennett,MatthewA;Shern,JackF;Kahn,RichardA
酵母双杂交系统的使用通过识别和表征新型蛋白质-蛋白质相互作用为许多以前未表征的途径提供了定义。双杂交系统使用转录因子的双功能性质，例如酵母增强子Gal 4，以允许通过报告基因转录的变化来监测蛋白质-蛋白质相互作用。一旦确定了积极的相互作用，可以通过位点特异性或随机引入的变化使相互作用蛋白质中的任一种突变，以产生相互作用能力降低的蛋白质。使用这种策略产生的突变体在测试相互作用的生物学意义和确定参与相互作用的残基方面是非常强大的试剂。这种技术被称为反向双杂交方法。我们描述了一种反向双杂交方法，该方法产生了大肠杆菌不耐热毒素（LTA 1）的催化亚基的相互作用缺失突变，其与活性（GTP结合）形式的人ARF 3（其蛋白辅因子）的结合减少。虽然出现了在哺乳动物细胞而不是酵母中进行相互作用筛选的新方法，但在酵母中使用反向双杂交仍然是鉴定失去相互作用点突变体和补偿变化的强大而有力的手段，这些突变体仍然是测试蛋白质-蛋白质相互作用的生物学意义的最有力的工具之一。
Use of the yeast two-hybrid system has provided definition to many previously uncharacterized pathways through the identification and characterization of novel protein-protein interactions. The two-hybrid system uses the bifunctional nature of transcription factors, such as the yeast enhancer Gal4, to allow protein-protein interactions to be monitored through changes in transcription of reporter genes. Once a positive interaction has been identified, either of the interacting proteins can be mutated by site-specific or randomly introduced changes, to produce proteins with a decreased ability to interact. Mutants generated using this strategy are very powerful reagents in tests of the biological significance of the interaction and in defining the residues involved in the interaction. Such techniques are termed reverse two-hybrid methods. We describe a reverse two-hybrid method that generates loss-of-interaction mutations of the catalytic subunit of theEscherichia coliheat-labile toxin (LTA1) with decreased binding to the active (GTP-bound) form of human ARF3, its protein cofactor. While newer methods are emerging for performing interaction screens in mammalian cells, instead of yeast, the use of reverse two-hybrid in yeast remains a robust and powerful means of identifying loss-of-interaction point mutants and compensating changes that remain among the most powerful tools of testing the biological significance of a protein-protein interaction.