USE OF POLYMERASE CHAIN-REACTION EPITOPE TAGGING FOR PROTEIN TAGGING IN SACCHAROMYCES-CEREVISIAE
USE OF POLYMERASE CHAIN-REACTION EPITOPE TAGGING FOR PROTEIN TAGGING IN SACCHAROMYCES-CEREVISIAE
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DOI：
10.1002/yea.320111306
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发表时间：
1995-10-01
期刊：
影响因子：
2.6
通讯作者：
FUTCHER, AB
中科院分区：
文献类型：
--
作者：
SCHNEIDER, BL;SEUFERT, W;FUTCHER, AB
表位标记是将构成表位的一小段氨基酸插入另一种蛋白质中。标记的蛋白质可以通过Western、免疫沉淀和免疫荧光测定使用预先存在的抗体来鉴定。我们设计了含有URA 3基因的载体，该基因两侧是表位标签的直接重复序列。我们使用聚合酶链反应（PCR）扩增tag-URA 3-tag盒，使得PCR片段的末端与感兴趣的基因具有同源性。然后使用体内重组将片段直接整合到目的位置，并通过其Ura（+）表型选择转化体。最后，在5-氟乳清酸平板上选择Ura（-）细胞产生这样的细胞，其中重复表位之间的重组已经“弹出"URA 3基因，在所需位置留下单拷贝的表位。PCR表位标记（PET）提供了一种快速和直接的标记技术，不需要任何克隆步骤。我们已经使用PET标记三个酿酒酵母蛋白，Cln 1，Sic 1和Est 1。
Epitope tagging is the insertion of a short stretch of amino acids constituting an epitope into another protein. Tagged proteins can be identified by Western, immunoprecipitation and immunofluorescence assays using pre-existing antibodies. We have designed vectors containing the URA3 gene flanked by direct repeats of epitope tags. We use the polymerase chain reaction (PCR) to amplify the tag-URA3-tag cassette such that the ends of the PCR fragments possess homology to the gene of interest. In vivo recombination is then used to direct integration of the fragment to the location of interest, and transformants are selected by their Ura(+) phenotype. Finally, selection for Ura(-) cells on 5-fluoro-orotic acid plates yields cells where recombination between the repeated epitopes has 'popped out' the URA3 gene, leaving a single copy of the epitope at the desired location. PCR epitope tagging (PET) provides a rapid and direct technique for tagging that does not require any cloning steps. We have used PET to tag three Saccharomyces cerevisiae proteins, Cln1, Sic1 and Est1.