通过基于标准Gal4p的酵母双杂交筛选鉴定蛋白质-蛋白质相互作用。

Identification of protein-protein interactions by standard gal4p-based yeast two-hybrid screening.

作者信息

Wagemans Jeroen, Lavigne Rob

机构信息

Laboratory of Gene Technology, Katholieke Universiteit Leuven, Kasteelpark Arenberg 21, 2462, 3001, Leuven, Belgium.

出版信息

Methods Mol Biol. 2015;1278:409-31. doi: 10.1007/978-1-4939-2425-7_27.

DOI:10.1007/978-1-4939-2425-7_27

PMID:25859966

Abstract

Yeast two-hybrid (Y2H) screening permits identification of completely new protein interaction partners for a protein of interest, in addition to confirming binary protein-protein interactions. After discussing the general advantages and drawbacks of Y2H and existing alternatives, this chapter provides a detailed protocol for traditional Gal4p-based Y2H library screens in Saccharomyces cerevisiae AH109. This includes bait transformation, bait auto-activation testing, prey library transformation, Y2H evaluation, and subsequent identification of the prey plasmids. Moreover, a one-on-one mating protocol to confirm interactions between suspected partners is given. Finally, a quantitative α-galactosidase assay protocol to compare interaction strengths is provided.

摘要

酵母双杂交（Y2H）筛选除了能确认二元蛋白质-蛋白质相互作用外，还能鉴定出感兴趣蛋白质全新的蛋白质相互作用伙伴。在讨论了Y2H及现有替代方法的一般优缺点后，本章提供了在酿酒酵母AH109中基于传统Gal4p的Y2H文库筛选的详细方案。这包括诱饵转化、诱饵自激活测试、猎物文库转化、Y2H评估以及随后猎物质粒的鉴定。此外，还给出了用于确认疑似伙伴之间相互作用的一对一交配方案。最后，提供了一种用于比较相互作用强度的定量α-半乳糖苷酶测定方案。