JCS：厦门大学靳全文教授课题组发表GFP结合蛋白相关研究论文

2017年3月1日，国际学术期刊《Journal of Cell science》杂志在线发表了厦门大学生命科学学院靳全文教授课题组题为《Facile manipulation of protein localization in fission yeast through binding of GFP-binding protein to GFP》的研究论文。陈应辉为该论文的第一作者，靳全文教授和王亚梅副教授为论文通讯作者。

GFP结合蛋白(GFP binding protein, GBP)是由美洲鸵单链抗体改造而得到的，对GFP有较高亲和性。在多种生物实验体系中，人们已经开始利用GBP富集带有GFP标签的蛋白，十分有效。GBP的分子质量较小，只有13 kDa，这有效地降低了对与之融合的蛋白功能的影响。通过GBP与GFP的高亲和力，可以有效地将目的蛋白强制定位到细胞特定的区域，人为改变或操控目的蛋白在细胞内的定位，帮助对目的基因的功能研究。

为了拓宽GBP-GFP系统在模式生物裂殖酵母中的应用，研究人员构建了一系列带有不同类型及不同强度启动子的质粒共计30个，可以用于整合到酵母基因组内表达融合了GBP或者GBP-mChery的目的蛋白。

在这项研究中，研究人员通过给纺锤体检验点蛋白Dma1及细胞胞质分裂起始网络激酶Cdc7添加GBP或者GBP-mChery标签，验证了所构建的质粒的有效性。通过GBP-GFP系统，Dma1可有效地被分别强制定位到细胞核膜、核基质、核仁、细胞端部以及胞质分裂环的结点。而通过Sid4-GFP将Cdc7-GBP提早募集到纺锤体极体SPB上时， 细胞胞质分裂信号通路被提前激活，细胞出现严重多隔和致死效应。这些结果显示，GBP/GBP-mChery标记的蛋白可以有效地被GFP标记的蛋白牵拉，这将方便人们对目的基因在特定细胞内区域的功能的研究。

原文链接：

Facile manipulation of protein localization in fission yeast through binding of gfp-binding protein to GFP

原文摘要：

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 yeastSchizosaccharomyces 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 constitutiveadh1 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.

doi:10.1242/jcs.198457

作者：靳全文 点击：次