A method for labeling proteins with tags at the native genomic loci in budding yeast.
A method for labeling proteins with tags at the native genomic loci in budding yeast.
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一种在芽殖酵母天然基因组位点用标签标记蛋白质的方法
DOI：
10.1371/journal.pone.0176184
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Fu YV
中科院分区：
文献类型：
--
作者：
Wang Q;Xue H;Li S;Chen Y;Tian X;Xu X;Xiao W;Fu YV
荧光蛋白和表位标签通常用作蛋白质融合标签来研究靶蛋白。芽殖酵母酿酒酵母中的一种流行技术是通过同源重组将这些标签融合到精确染色体位置的靶基因上。然而，一些限制阻碍了该技术的应用，例如选择性标记不可重复使用，仅标记C-末端是可能的，以及在基因组中留下“疤痕”序列。在这里，我们描述了一种策略来解决这些问题，标记靶基因的基础上弹出/弹出和反选择系统。开发了三种荧光蛋白标签（mCherry、sfGFP和mKikGR）和两种表位标签（HA和3×FLAG）构建体，并用于在染色体基因座处标记HHT 1、UBC 13或RAD 5作为概念验证。
Fluorescent proteins and epitope tags are often used as protein fusion tags to study target proteins. One prevailing technique in the budding yeast Saccharomyces cerevisiae is to fuse these tags to a target gene at the precise chromosomal location via homologous recombination. However, several limitations hamper the application of this technique, such as the selectable markers not being reusable, tagging of only the C-terminal being possible, and a “scar” sequence being left in the genome. Here, we describe a strategy to solve these problems by tagging target genes based on a pop-in/pop-out and counter-selection system. Three fluorescent protein tag (mCherry, sfGFP, and mKikGR) and two epitope tag (HA and 3×FLAG) constructs were developed and utilized to tag HHT1, UBC13 or RAD5 at the chromosomal locus as proof-of-concept.