A vector system for efficient and economical switching of C-terminal epitope tags in Saccharomyces cerevisiae
A vector system for efficient and economical switching of C-terminal epitope tags in Saccharomyces cerevisiae
复制标题
DOI：
10.1002/yea.1588
复制
发表时间：
2008-04-01
期刊：
影响因子：
2.6
通讯作者：
Huh, Won-Ki
中科院分区：
文献类型：
--
作者：
Sung, Min-Kyung;Ha, Cheol Woong;Huh, Won-Ki
在酿酒酵母（Saccharomyces cerevisiae）中，一步pcr介导的染色体基因修饰允许在C端或n端快速有效地标记酵母蛋白的各种表位。在许多用途上，c末端标记的优势在于表位标签的表达模式与真实蛋白的表达模式相当，并且在翻译过程中标签影响多肽链正常折叠的可能性最小。随着实验的复杂化，往往需要构建多个带有不同表位标记的融合蛋白。在这里，我们描述了一系列质粒的发展，这些质粒仅使用一组通用寡核苷酸引物就可以高效经济地切换c末端标记的表位。该质粒含有多种表位（GFP、TAP、GST、Myc、HA和FLAG标签）和作为选择性标记的klluyveromyces laactis URA3基因，可用于用6个表位中的任意一个替换基于MX6模块的C-terminall表位标签。此外，质粒还允许对已经携带MX6模块基因缺失或c -末端表位标签的酵母细胞中的蛋白质进行额外的c -末端表位标记。版权所有(c) 2008 John Wiley & Sons Ltd。
In Saccharomyces cerevisiae, one-step PCR-mediated modification of chromosomal genes allows fast and efficient tagging of yeast proteins with various epitopes at the C- or N-terminus. For many purposes, C-terminall tagging is advantageous in that the expression pattern of epitope tag is comparable to that of the authentic protein and the possibility for the tag to affect normal folding of polypeptide chain during translation is minimized. As experiments are getting complicated, it is often necessary to construct several fusion proteins tagged with various kinds of epitopes. Here, we describe development of a series of plasmids that allow efficient and economical switching of C-terminally tagged epitopes, using just one set of universal oligonucleotide primers. Containing a variety of epitopes (GFP, TAP, GST, Myc, HA and FLAG tag) and Kluyveromyces lactis URA3 gene as a selectable marker, the plasmids can be used to replace any MX6 module-based C-terminall epitope tag with one of the six epitopes. Furthermore, the plasmids also allow additional C-terminal epitope tagging of proteins in yeast cells that already carry MX6 module-based gene deletion or C-terminal epitope tag. Copyright (c) 2008 John Wiley & Sons Ltd,.