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用于酿酒酵母中基于PCR的C端标记的小表位-接头模块

Small epitope-linker modules for PCR-based C-terminal tagging in Saccharomyces cerevisiae.

作者信息

Funakoshi Minoru, Hochstrasser Mark

机构信息

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.

出版信息

Yeast. 2009 Mar;26(3):185-92. doi: 10.1002/yea.1658.

Abstract

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 x 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).

摘要

PCR介导的基因修饰是对酿酒酵母基因进行功能分析的一种强大方法。该方法的一个应用是对基因进行表位标签,以便通过免疫学方法分析相应的蛋白质。然而,以方便形式提供的表位标签数量仍然很少,而且表位对蛋白质功能的干扰,特别是如果它很大的话,并不罕见。为了解决这些局限性并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末端标签,带有10种不同的、相对较短的肽序列,这些序列可被市售单克隆抗体识别。编码的标签有FLAG、3×FLAG、T7、His标签、Strep-tag II、S-tag、Myc、HSV、VSV-G和V5。同一对引物可用于构建带有10种标签中任何一种的感兴趣基因的标签等位基因。此外,在这些标签的上游插入了一个六甘氨酸接头序列,以尽量减少标签对靶蛋白的影响,并最大限度地提高其与抗体结合的可及性。每个表位有三个标记基因,即HIS3MX6、kanMX6和hphMX4。我们展示了这些新标签在免疫印迹和26S蛋白酶体调节颗粒的一步亲和纯化中的实用性。该质粒集已保存在非营利性质粒储存库Addgene(http://www.addgene.org)中。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/06f9/2673350/8bb25045912e/yea0026-0185-f1.jpg

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