The reverse two-hybrid system: A genetic scheme for selection against specific protein/protein interactions
The reverse two-hybrid system: A genetic scheme for selection against specific protein/protein interactions
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DOI：
10.1093/nar/24.17.3341
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发表时间：
1996-09-01
影响因子：
14.9
通讯作者：
Hannink, M
中科院分区：
文献类型：
--
作者：
Leanna, CA;Hannink, M
酵母双杂交系统是表征蛋白质/蛋白质相互作用的一种强有力的实验方法。酵母双杂交系统的一个独特优势是提供了基因选择技术，使之能够识别特定的蛋白质/蛋白质相互作用。我们现在报告了一种改进的酵母双杂交系统的开发，该系统能够针对特定的蛋白质/蛋白质相互作用进行遗传选择。这种反向双杂交系统利用了一种酵母菌株，由于存在突变的cyh2基因，该菌株对放线菌素具有抗药性。该菌株还含有Gall启动子转录控制下的野生型CYH2等位基因。野生型Gal4蛋白的表达足以恢复对放线菌素的生长敏感性，禽类c-rel蛋白与其I kappa Bα蛋白p40作为Gal4融合蛋白的共表达也恢复了对放线菌素的生长敏感性。恢复对放线菌胺的生长敏感性需要c-rel和p40在Gall启动子上的结合，并与c-rel/p40相互作用激活Gall启动子表达的能力有关。针对特定蛋白质/蛋白质相互作用的遗传选择方案可能是分析蛋白质/蛋白质相互作用的有价值的工具。
The yeast two-hybrid system is a powerful experimental approach for the characterization of protein/ protein interactions. A unique strength of the yeast two-hybrid system is the provision for genetic selection techniques that enable the identification of specific protein/protein interactions. We now report the development of a modified yeast two-hybrid system which enables genetic selection against a specific protein/protein interaction. This reverse two-hybrid system utilizes a yeast strain which is resistant to cycloheximide due to the presence of a mutant cyh2 gene. This strain also contains the wild-type CYH2 allele under the transcriptional control of the Gall promoter. Expression of the wild-type Gal4 protein is sufficient to restore growth sensitivity to cycloheximide, Growth sensitivity towards cycloheximide is also restored by the coexpression of the avian c-Rel protein and its I kappa B alpha counterpart, p40, as Gal4 fusion proteins. Restoration of growth sensitivity towards cycloheximide requires the association of c-Rel and p40 at the Gall promoter and correlates with the ability of the c-Rel/p40 interaction to activate expression from the Gall promoter. A genetic selection scheme against specific protein/protein interactions may be a valuable tool for the analysis of protein/protein interactions.