Engineering a Novel Multifunctional Green Fluorescent Protein Tag for a Wide Variety of Protein Research

Takuya Kobayashi,Taku Kashiyama,Hideto Oyamada,Nagomi Kurebayashi,Takashi Murayama,Nobuhiro Morone,Axel Imhof

doi:10.1371/journal.pone.0003822

Takuya Kobayashi, Taku Kashiyama + Show 5 more

Open Access

https://doi.org/10.1371/journal.pone.0003822

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Abstract

BackgroundGenetically 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 FindingsHere we report a novel multifunctional green fluorescent protein (mfGFP) tag which was engineered by inserting multiple peptide tags, i.e., octa-histidine (8×His), 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 8×His 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 SignificanceThe 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.

Highlights

Protein complexes mediate the majority of cellular processes
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
The Asp173-Gly174 was chosen as an insertion site, because it is located at the opposite side of N- and Ctermini that would allow tags to work effectively by minimizing steric hindrance provided by the fusion partner (Fig. 1A)

Summary

Introduction

Protein complexes mediate the majority of cellular processes. Information on localization, structure, and interaction of such protein complexes provides key insights into their functions. The use of multiple tags, such as tandem affinity purification (TAP) strategy, has recently become popular for purification of the protein complexes with a high purity [6]. Epitope tags are useful for affinity isolation of the protein complexes by immunoprecipitation.

Results

Conclusion