酿酒酵母中基于TurboID的邻近标记系统构建与应用
Construction and Application of a TurboID-Based Proximity Labeling System in Saccharomyces cerevisiae
刘艳 1高强1
作者信息
- 1. 湖南大学生命医学交叉研究院，长沙 410082
- 折叠
摘要
为实现在活细胞中对小分子量膜蛋白邻近环境及其相互作用网络进行高时空分辨率解析，本研究在酿酒酵母中构建了基于TurboID的邻近标记体系。以小蛋白Sna1/2p为模型，在其C末端融合TurboID，通过免疫印迹检测蛋白的表达及其对邻近蛋白的生物素化能力。结果表明，Sna1/2-TurboID在细胞内能成功表达，并有效催化邻近蛋白发生生物素化修饰现象。此外，为进一步降低细胞中本底生物素化水平的干扰，我们又敲除了内源性生物素化蛋白Arc1p，发现确实能一定程度降低背景信号。随后，通过时间梯度优化，确定3h为最佳标记时间。将细胞进行处理并经链霉亲和素磁珠富集后，样品可用于后续质谱分析。综上所述，本研究建立了一套适用于酿酒酵母中膜定位小分子量蛋白互作研究的邻近标记系统，为该类蛋白的动态互作网络分析提供了有效工具。
Abstract
This work developed a TurboID-based proximity labeling system in Saccharomyces cerevisiae to obtain high spatiotemporal resolution investigation of the local environment and interaction networks of small-molecular-weight membrane proteins in living cells. TurboID was fused to the C-terminus of the small protein Sna1/2p as a model, and Western blotting was utilized to identify protein expression and its capacity to biotinylate nearby proteins. The findings show that Sna1/2-TurboID efficiently catalyzes the biotinylation of nearby proteins and is successfully produced intracellularly. Additionally, we eliminated the endogenous biotinylated protein Arc1p in order to further limit interference from background biotinylation levels in cells. We discovered that this did, in fact, somewhat lower background signals.Three hours was then found to be the ideal labeling time using time-course optimization. The samples were prepared for further mass spectrometry analysis after the cells were treated and enriched using streptavidin magnetic beads. In conclusion, our work developed a proximity labeling technique that may be used to analyze the dynamic interaction networks of membrane-localized small-molecular-weight proteins in Saccharomyces cerevisiae.关键词
酿酒酵母/邻近标记技术/TurboIDKey words
Saccharomyces cerevisiae/proximity labeling technology/TurboID引用本文复制引用
刘艳,高强.酿酒酵母中基于TurboID的邻近标记系统构建与应用[EB/OL].(2026-03-26)[2026-03-27].http://www.paper.edu.cn/releasepaper/content/202603-260.学科分类
生物科学研究方法、生物科学研究技术
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