Engineering a Novel Multifunctional Green Fluorescent Protein Tag for a Wide Variety of Protein Research Takuya Kobayashi 1 , Nobuhiro Morone 2 , Taku Kashiyama 1 , Hideto Oyamada 3 , Nagomi Kurebayashi 1 , Takashi Murayama 1 * 1 Department of Pharmacology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, Japan, 2 Department of Ultrastructural Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan, 3 Department of Pharmacology, School of Medicine, Showa University, Shinagawa-ku, Tokyo, Japan Abstract 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 (8 6 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 6 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 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. Citation: Kobayashi T, Morone N, Kashiyama T, Oyamada H, Kurebayashi N, et al. (2008) Engineering a Novel Multifunctional Green Fluorescent Protein Tag for a Wide Variety of Protein Research. PLoS ONE 3(12): e3822. doi:10.1371/journal.pone.0003822 Editor: Axel Imhof, University of Munich and Center of Integrated Protein Science, Germany Received July 14, 2008; Accepted November 7, 2008; Published December 2, 2008 Copyright: ß 2008 Kobayashi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by grants from the Japan Science and Technology Agency (SENTAN), Grants-in-Aid for Scientific Research, Japan Society for Promotion of Science, and a Health Labor Sciences Research Grant (NANO-001). Competing Interests: The authors have declared that no competing interests exist. * E-mail: takashim@juntendo.ac.jp 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. Genetically encoded protein tags are a useful tool for character- ization of protein complexes. There are a variety of tags for localizing proteins in living cells. Among them, the most widely used are Aequorea victoria green fluorescent protein (GFP) and the related fluorescent proteins, which are easily fused to proteins of interest [1,2]. Because the N- and C-termini of GFP are closely located to each other, this protein can be inserted in the middle of the fusion partner, as well as fusion at its N- and C-termini [3]. This is great advantage in case that the protein has intolerant of tag fusion at both N- and C-termini. Structural and interaction analysis of the protein complexes requires isolation of the complex. A variety of affinity tags have been developed, which strongly bind to a specified ligand [4,5]. 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, small peptides to which commercially available highly specific antibody bind, are used for a wide variety of immunological detections [7]. Epitope tags are also useful for affinity isolation of the protein complexes by immunoprecipitation. In general, cellular localization and isolation of protein complexes are determined using fusion proteins with different tags; GFP-tagged protein for localization and affinity tagged- protein for structural and protein composition analysis. However, making of different constructs is time- and resource-consuming, especially in case of analyzing a large number of proteins, e.g., genome-wide analysis. In addition, it is better to correlate the two determinations using the same fusion protein. Development of a single tag with multifunction is therefore highly desirable. GFP forms a rigid and stable 11-stranded b-barrel structures [1,2] (see Fig. 1A). It has been shown that GFP is tolerant to insertion of foreign peptides within certain loops between the b- strands [8] and thus can be a scaffold for short peptides [9,10]. In this study, we engineered ‘‘multifunctional GFP’’ (mfGFP), in which the multiple peptide tags (affinity tags and epitope tags) were PLoS ONE | www.plosone.org 1 December 2008 | Volume 3 | Issue 12 | e3822