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下一代酵母双混合查以发现蛋白质-蛋白质相互作用

J Mitch Elmore^1,2,3, Valeria Velásquez-Zapata^4,5, Roger P Wise^6,4,5

¹USDA-Agricultural Research Service, Cereal Disease Laboratory, St. Paul, MN, USA. mitch.elmore@usda.gov.

Methods in molecular biology (Clifton, N.J.)

|July 14, 2023

概括

这项研究引入了一种精简的酵母双混合选方法,使用批量液体培养. 这种方法通过下一代测序有效地识别蛋白质-蛋白质相互作用,增强新生物伙伴关系的发现.

科学领域:

分子生物学分子生物学
生物化学生物化学
遗传学是一个遗传学.

背景情况:

酵母二混合 (Y2H) 是确定蛋白质与蛋白质相互作用的关键方法.
传统的Y2H查需要单个殖民地分析,这很费力和耗时.
开发高通量Y2H选方法对于推进互原子映射至关重要.

研究的目的:

提出一个简化的,基于批量的酵母两混合查协议.
从大型cDNA库中有效识别和量化蛋白质-蛋白质相互作用.
为了利用下一代测序来对Y2H选结果进行高通量分析.

主要方法:

在批量液体培养中实施酵母双混合选.
在选择性压力下丰富阳性酵母细胞种群.
使用下一代测序来放大和分析猎物cDNAs.

主要成果:

使用批量液体培养Y2H方法成功识别了蛋白质与蛋白质相互作用.
通过下一代测序来量化和排列相互作用的猎物cDNA.
简化和高效的Y2H选工作流程的演示.

结论:

描述的批量液体培养Y2H方法可显著提高效率和吞吐量.
这种方法有助于大规模发现蛋白质与蛋白质相互作用.
与下一代测序的整合为交互原子研究提供了一个强大的工具.

关键词:

互动查对相互作用进行查.蛋白质蛋白质相互作用序列化是指测序的使用.酵母两种混合的酵母在cDNA图书馆里.

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