Engineering a Novel Multifunctional Green Fluorescent Protein Tag for a Wide Variety of Protein Research
Engineering a Novel Multifunctional Green Fluorescent Protein Tag for a Wide Variety of Protein Research
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DOI：
10.1371/journal.pone.0003822
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发表时间：
2008-12-02
期刊：
影响因子：
3.7
通讯作者：
Murayama, Takashi
中科院分区：
文献类型：
--
作者：
Kobayashi, Takuya;Morone, Nobuhiro;Murayama, Takashi
背景：基因编码标签是蛋白质研究的有力工具。各种标记已经被开发出来：用于活细胞成像的荧光蛋白，用于蛋白质分离的亲和标记，以及用于免疫学检测的表位标记。蛋白质标记的主要问题之一是需要为不同的应用制作许多不同标签的结构，这既耗时又耗费资源。方法/主要发现：本文报道了一种新型的多功能绿色荧光蛋白(MfGFP)标签，它是通过将八个组氨酸(86His)、链霉亲和素结合肽(SBP)和c-Myc标签串联插入到GFP的循环中而设计的。当mfGFP与不同的蛋白质融合时，mfGFP监测它们在活细胞中的定位。带有SBP标签的链霉亲和素琼脂糖柱层析成功地分离出天然形式的高纯度蛋白质复合体。MfGFP中带有8xHis和SBP标签的串联亲和纯化(TAP)进一步纯化了蛋白质复合体。在免疫荧光和免疫电子显微镜(EM)中，c-Myc特异性抗体均能清晰地检测到mfGFP。这些发现表明，mfGFP在哺乳动物细胞中作为多功能标签发挥了很好的作用。结论和意义：多功能荧光蛋白标签是一种用于多种蛋白质研究的有用工具，与其他多重标签系统相比具有更高的可扩展性和与现有和未来的标签技术兼容的优势。
Background: Genetically encoded tag is a powerful tool for protein research. Various kinds of tags have been developed: fluorescent proteins for live-cell imaging, affinity tags for protein isolation, and epitope tags for immunological detections. One of the major problems concerning the protein tagging is that many constructs with different tags have to be made for different applications, which is time- and resource-consuming.Methodology/Principal Findings: Here we report a novel multifunctional green fluorescent protein (mfGFP) tag which was engineered by inserting multiple peptide tags, i.e., octa-histidine (86His), streptavidin-binding peptide (SBP), and c-Myc tag, in tandem into a loop of GFP. When fused to various proteins, mfGFP monitored their localization in living cells. Streptavidin agarose column chromatography with the SBP tag successfully isolated the protein complexes in a native form with a high purity. Tandem affinity purification (TAP) with 8xHis and SBP tags in mfGFP further purified the protein complexes. mfGFP was clearly detected by c-Myc-specific antibody both in immunofluorescence and immuno-electron microscopy (EM). These findings indicate that mfGFP works well as a multifunctional tag in mammalian cells. The tag insertion was also successful in other fluorescent protein, mCherry.Conclusions and Significance: The multifunctional fluorescent protein tag is a useful tool for a wide variety of protein research, and may have the advantage over other multiple tag systems in its higher expandability and compatibility with existing and future tag technologies.