一种改进的酿酒酵母基因串联亲和纯化标记策略。
An improved strategy for tandem affinity purification-tagging of Schizosaccharomyces pombe genes.
机构信息
Max F. Perutz Laboratories, University of Vienna, Vienna, Austria.
出版信息
Proteomics. 2009 Oct;9(20):4825-8. doi: 10.1002/pmic.200800948.
Abstract
Tandem affinity purification (TAP) is a method that allows rapid purification of native protein complexes. We developed an improved technique to fuse the fission yeast genes with a TAP tag. Our technique is based on tagging constructs that contain regions homologous to the target gene cloned into vectors carrying a TAP tag. We used this technique to design strategies for TAP-tagging of predicted Schizosaccharomyces pombe genes (http://mendel.imp.ac.at/Pombe_tagging/). To validate the approach, we purified the proteins, which associated with two evolutionarily conserved proteins Swi5 and Sfr1 as well as three protein kinases Ksg1, Orb6 and Sid1.
摘要
串联亲和纯化（TAP）是一种能够快速纯化天然蛋白复合物的方法。我们开发了一种改良技术，可将裂殖酵母基因与 TAP 标签融合。我们的技术基于标记构建体，该构建体包含与目标基因克隆到携带 TAP 标签的载体中的同源区域。我们使用该技术设计了用于 TAP 标记预测的酿酒酵母基因的策略（http://mendel.imp.ac.at/Pombe_tagging/）。为了验证该方法，我们纯化了与两个进化上保守的蛋白质 Swi5 和 Sfr1 以及三个蛋白激酶 Ksg1、Orb6 和 Sid1 相关的蛋白质。
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1
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Genetics. 2008 Dec;180(4):1859-68. doi: 10.1534/genetics.108.093005. Epub 2008 Oct 9.
2
Epitope tagging of endogenous genes in diverse human cell lines.在多种人类细胞系中对内源基因进行表位标记
Nucleic Acids Res. 2008 Nov;36(19):e127. doi: 10.1093/nar/gkn566. Epub 2008 Sep 10.
3
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Curr Genet. 2008 Oct;54(4):197-211. doi: 10.1007/s00294-008-0212-z. Epub 2008 Sep 4.
4
Reconstitution of DNA strand exchange mediated by Rhp51 recombinase and two mediators.由Rhp51重组酶和两种介导因子介导的DNA链交换的重建
PLoS Biol. 2008 Apr 15;6(4):e88. doi: 10.1371/journal.pbio.0060088.
5
Formation and branch migration of Holliday junctions mediated by eukaryotic recombinases.真核生物重组酶介导的霍利迪连接体的形成与分支迁移
Nature. 2008 Feb 21;451(7181):1018-21. doi: 10.1038/nature06609. Epub 2008 Feb 6.
6
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7
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9
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