Small epitope-linker modules for PCR-based C-terminal tagging in Saccharomyces cerevisiae.
Small epitope-linker modules for PCR-based C-terminal tagging in Saccharomyces cerevisiae.
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DOI：
10.1002/yea.1658
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发表时间：
2009-03
期刊：
影响因子：
2.6
通讯作者：
Hochstrasser, Mark
中科院分区：
文献类型：
--
作者：
Funakoshi, Minoru;Hochstrasser, Mark
PCR介导的基因修饰是研究酿酒酵母基因功能的有效方法。该方法的一个应用是通过免疫学方法对基因进行表位标记以分析相应的蛋白质。然而，以方便的形式可用的表位标签的数量仍然很低，并且表位对蛋白质功能的干扰，特别是如果它很大，并不罕见。为了解决这些限制和扩大该方法的实用性，我们构建了一组方便的模板质粒，设计用于基于PCR的C-末端标记，具有10种不同的、相对较短的肽序列，这些肽序列可被市售单克隆抗体识别。编码的标签是FLAG、3 × FLAG、T7、His-标签、Strep-标签II、S-标签、Myc、HSV、VSV-G和V5。相同的引物对可用于构建具有10个标签中的任一个的感兴趣基因的标记等位基因。此外，将六甘氨酸接头序列插入这些标签的上游，以使标签对靶蛋白的影响最小化，并使其与抗体结合的可及性最大化。三种标记基因，HIS 3 MX6、kanMX 6和hphMX 4，可用于每个表位。我们证明了新标签的实用性，用于26 S蛋白酶体的调节颗粒的免疫印迹和一步亲和纯化。这组质粒已保藏在非营利质粒库Addgene（http：//www.example.com）中。www.addgene.org
PCR-mediated gene modification is a powerful approach to the functional analysis of genes in Saccharomyces cerevisiae. One application of this method is epitope-tagging of a gene to analyse the corresponding protein by immunological methods. However, the number of epitope tags available in a convenient format is still low, and interference with protein function by the epitope, particularly if it is large, is not uncommon. To address these limitations and broaden the utility of the method, we constructed a set of convenient template plasmids designed for PCR-based C-terminal tagging with 10 different, relatively short peptide sequences that are recognized by commercially available monoclonal antibodies. The encoded tags are FLAG, 3 × FLAG, T7, His-tag, Strep-tag II, S-tag, Myc, HSV, VSV-G and V5. The same pair of primers can be used to construct tagged alleles of a gene of interest with any of the 10 tags. In addition, a six-glycine linker sequence is inserted upstream of these tags to minimize the influence of the tag on the target protein and maximize its accessibility for antibody binding. Three marker genes, HIS3MX6, kanMX6 and hphMX4, are available for each epitope. We demonstrate the utility of the new tags for both immunoblotting and one-step affinity purification of the regulatory particle of the 26S proteasome. The set of plasmids has been deposited in the non-profit plasmid repository Addgene (http://www.addgene.org).
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