Facile manipulation of protein localization in fission yeast through binding of GFP-binding protein to GFP
Facile manipulation of protein localization in fission yeast through binding of GFP-binding protein to GFP
复制标题
通过 GFP 结合蛋白与 GFP 的结合轻松操纵裂殖酵母中的蛋白质定位
DOI：
10.1242/jcs.198457
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发表时间：
2017-03-01
影响因子：
4
通讯作者：
Jin, Quan-wen
中科院分区：
文献类型：
--
作者：
Chen, Ying-hui;Wang, Gao-yuan;Jin, Quan-wen
摘要 GFP 结合蛋白（或 GBP）最近在各种系统和生物体中被开发为纯化 GFP 融合蛋白的有效工具。由于 GBP 和 GFP 或 GFP 变体之间的高亲和力，这种基于 GBP 的方法也非常适合改变活细胞中功能蛋白的定位。为了促进 GBP 靶向方法在裂殖酵母裂殖酵母中的广泛使用，我们开发了一组基于 pFA6a、pJK148 和 pUC119 的载体，其中包含 GBP 或 GBP–mCherry 编码序列以及诱导型 nmt1 或组成型 adh1 启动子的变体，从而产生不同的表达水平。 GBP 或 GBP-mCherry 片段可用作感兴趣基因的 N 端或 C 端基因组标签的盒。我们说明了这些载体在构建带有 GBP-mCherry 标记的 Dma1 或 Cdc7 的酵母菌株中的应用，以及通过 Sid4-GFP 将 Dma1- 或 Cdc7-GBP-mCherry 有效靶向纺锤体极体。这一系列载体应有助于促进 GBP 靶向方法在单基因水平以及系统规模上操纵蛋白质定位和裂殖酵母基因功能分析中的应用。摘要：构建了一组包含 GBP- 或 GBP-mCherry 编码序列以及诱导型 nmt1 或组成型 adh1 启动子变体的载体。它们应该允许更容易地人工操作裂殖酵母中的蛋白质定位。
ABSTRACT GFP-binding protein (or GBP) has been recently developed in various systems and organisms as an efficient tool to purify GFP-fusion proteins. Due to the high affinity between GBP and GFP or GFP variants, this GBP-based approach is also ideally suited to alter the localization of functional proteins in live cells. In order to facilitate the wide use of the GBP-targeting approach in the fission yeast Schizosaccharomyces pombe, we developed a set of pFA6a-, pJK148- and pUC119-based vectors containing GBP- or GBP–mCherry-coding sequences and variants of inducible nmt1 or constitutive adh1 promoters that result in different levels of expression. The GBP or GBP–mCherry fragments can serve as cassettes for N- or C-terminal genomic tagging of genes of interest. We illustrated the application of these vectors in the construction of yeast strains with Dma1 or Cdc7 tagged with GBP–mCherry and efficient targeting of Dma1– or Cdc7–GBP–mCherry to the spindle pole body by Sid4–GFP. This series of vectors should help to facilitate the application of the GBP-targeting approach in manipulating protein localization and the analysis of gene function in fission yeast, at the level of single genes, as well as at a systematic scale. Summary: A set of vectors containing GBP- or GBP–mCherry-coding sequences and variants of inducible nmt1 or constitutive adh1 promoters were constructed. They should allow easier artificial manipulation of protein localization in fission yeast.