A PCR-based strategy to generate yeast strains expressing endogenous levels of amino-terminal epitope-tagged proteins
A PCR-based strategy to generate yeast strains expressing endogenous levels of amino-terminal epitope-tagged proteins
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DOI：
10.1002/biot.200800012
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发表时间：
2008-04-01
影响因子：
4.7
通讯作者：
Kaiser, Peter
中科院分区：
文献类型：
--
作者：
Booher, Keith R.;Kaiser, Peter
引入感兴趣基因（GOI）的表位标签大大增加了研究细胞蛋白的方便性。基于快速聚合酶链反应的抗原表位标记策略在酵母中得到了广泛的应用。然而，c端表位标记并不适用于所有的蛋白质。融合到c末端的表位标签可以干扰某些蛋白质的功能，甚至可以通过c末端蛋白质加工去除。为了克服这些问题，可以在蛋白质的氨基末端用表位标记，但在天然位点的内源性启动子控制下，产生表达n端表位标记基因的酵母菌株相对困难。以前曾报道过引入n端表位标签的策略，但通常是在基因组中引入除表位标签以外的其他序列。此外，用目前的方法对必需基因进行n端标记需要形成二倍体菌株，然后进行四分体解剖或从质粒中表达额外的GOI拷贝。本文描述的策略提供了一种快速、简便的方法，在两步pcr的基础上标记必需和非必需基因的n端。该方法的显著优点是将标记的基因置于其内源性启动子的控制之下，并且除了编码核片段的表位标签外，没有其他序列插入基因组中。
An epitope tag introduced to a gene of interest (GOI) greatly increases the ease of studying cellular proteins. Rapid PCR-based strategies for epitope tagging a protein's C-terminus at its native gene locus are widely used in yeast. C-terminal epitope tagging is not suitable for all proteins, however. Epitope tags fused to the C-terminus can interfere with function of some proteins or can even be removed by C-terminal protein processing. To overcome such problems, proteins can be tagged with epitopes at their amino-termini, but generating yeast strains expressing N-terminal epitope tagged genes under control of the endogenous promoter at the native locus is comparatively more difficult. Strategies to introduce N-terminal epitope tags have been reported previously but often introduce additional sequences other than the epitope tag into the genome. Furthermore, N-terminal tagging of essential genes by current methods requires formation of diploid strains followed by tetrad dissection or expression of an additional copy of the GOI from a plasmid. The strategies described here provide a quick, facile means of epitope tagging the N-terminus of both essential and nonessential genes in a two-step PCR-based procedure. The procedure has the significant advantage of leaving tagged genes under the control of their endogenous promoters, and no additional sequences other than the epitope tag encoding nucleoticles are inserted into the genome.