Jiang Yuwei, Di Gregorio Sonja E, Duennwald Martin L, Lajoie Patrick
Department of Anatomy and Cell Biology, The University of Western Ontario, London, Canada.
Department of Pathology and Laboratory Medicine, The University of Western Ontario, London, Canada.
Traffic. 2017 Jan;18(1):58-70. doi: 10.1111/tra.12453. Epub 2016 Nov 1.
The palette of fluorescent proteins (FPs) available for live-cell imaging contains proteins that strongly differ in their biophysical properties. FPs cannot be assumed to be equivalent and in certain cases could significantly perturb the behavior of fluorescent reporters. We employed Saccharomyces cerevisiae to comprehensively study the impact of FPs on the toxicity of polyglutamine (polyQ) expansion proteins associated with Huntington's disease. The toxicity of polyQ fusion constructs is highly dependent on the sequences flanking the polyQ repeats. Thus, they represent a powerful tool to study the impact of fluorescent fusion partners. We observed significant differences on polyQ aggregation and toxicity between commonly used FPs. We generated a novel series of vectors with latest yeast-optimized FPs for investigation of Htt toxicity, including a newly optimized blue FP for expression in yeast. Our study highlights the importance of carefully choosing the optimal FPs when designing tagging strategies.
用于活细胞成像的荧光蛋白（FPs）库包含生物物理特性差异很大的蛋白质。不能认为荧光蛋白是等同的，在某些情况下，它们可能会显著干扰荧光报告基因的行为。我们利用酿酒酵母全面研究荧光蛋白对与亨廷顿舞蹈病相关的聚谷氨酰胺（polyQ）扩展蛋白毒性的影响。聚Q融合构建体的毒性高度依赖于聚Q重复序列两侧的序列。因此，它们是研究荧光融合伴侣影响的有力工具。我们观察到常用荧光蛋白在聚Q聚集和毒性方面存在显著差异。我们生成了一系列新型载体，带有最新的酵母优化荧光蛋白，用于研究亨廷顿蛋白（Htt）的毒性，包括一种新优化的蓝色荧光蛋白，可在酵母中表达。我们的研究强调了在设计标记策略时仔细选择最佳荧光蛋白的重要性。
