Gauss Robert, Trautwein Mark, Sommer Thomas, Spang Anne
Max-Delbrück Centrum für Molekulare Medizin, Robert-Rössle Str. 10, D-13092, Berlin, Germany.
Yeast. 2005 Jan 15;22(1):1-12. doi: 10.1002/yea.1187.
Epitope tagging is a powerful method for the rapid analysis of protein function. In Saccharomyces cerevisiae epitope tags are introduced easily into chromosomal loci by homologous recombination using a simple PCR-based strategy. Although quite a number of tools exist for C-terminal tagging as well as N-terminal tagging of proteins expressed by heterologous promoters, there are only very limited possibilities to tag proteins at the N-terminus and retain the endogenous expression level. Furthermore, no PCR-templates for internal tagging have been reported. Here we describe new modules that are suitable for both the repeated N-terminal and internal tagging of proteins, leaving their endogenous promoters intact. The tags include 6xHA, 9xMyc, yEGFP, TEV-GST-6xHIS, ProtA, TEV-ProtA and TEV-ProtA-7xHIS in conjunction with different heterologous selection markers.
表位标签法是一种用于快速分析蛋白质功能的强大方法。在酿酒酵母中，使用基于简单PCR的策略通过同源重组可轻松地将表位标签引入染色体位点。尽管存在许多用于异源启动子表达的蛋白质的C端标签以及N端标签的工具，但在N端对蛋白质进行标签并保持内源性表达水平的可能性非常有限。此外，尚未报道用于内部标签的PCR模板。在此，我们描述了适用于蛋白质重复N端和内部标签的新模块，同时保持其内源启动子完整。这些标签包括6xHA、9xMyc、yEGFP、TEV-GST-6xHIS、ProtA、TEV-ProtA和TEV-ProtA-7xHIS，并结合了不同的异源选择标记。
