Engineering a novel multifunctional green fluorescent protein tag for a wide variety of protein research.

<h4>Background</h4>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 concern...

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Autores principales:	Takuya Kobayashi, Nobuhiro Morone, Taku Kashiyama, Hideto Oyamada, Nagomi Kurebayashi, Takashi Murayama
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2008
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/2749fe4599ca462299c566b4b19ff582
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spelling	oai:doaj.org-article:2749fe4599ca462299c566b4b19ff5822021-11-25T06:18:21ZEngineering a novel multifunctional green fluorescent protein tag for a wide variety of protein research.1932-620310.1371/journal.pone.0003822https://doaj.org/article/2749fe4599ca462299c566b4b19ff5822008-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/19048102/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>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.<h4>Methodology/principal findings</h4>Here we report a novel multifunctional green fluorescent protein (mfGFP) tag which was engineered by inserting multiple peptide tags, i.e., octa-histidine (8xHis), 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.<h4>Conclusions and significance</h4>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.Takuya KobayashiNobuhiro MoroneTaku KashiyamaHideto OyamadaNagomi KurebayashiTakashi MurayamaPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 3, Iss 12, p e3822 (2008)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Takuya Kobayashi Nobuhiro Morone Taku Kashiyama Hideto Oyamada Nagomi Kurebayashi Takashi Murayama Engineering a novel multifunctional green fluorescent protein tag for a wide variety of protein research.
description	<h4>Background</h4>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.<h4>Methodology/principal findings</h4>Here we report a novel multifunctional green fluorescent protein (mfGFP) tag which was engineered by inserting multiple peptide tags, i.e., octa-histidine (8xHis), 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.<h4>Conclusions and significance</h4>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.
format	article
author	Takuya Kobayashi Nobuhiro Morone Taku Kashiyama Hideto Oyamada Nagomi Kurebayashi Takashi Murayama
author_facet	Takuya Kobayashi Nobuhiro Morone Taku Kashiyama Hideto Oyamada Nagomi Kurebayashi Takashi Murayama
author_sort	Takuya Kobayashi
title	Engineering a novel multifunctional green fluorescent protein tag for a wide variety of protein research.
title_short	Engineering a novel multifunctional green fluorescent protein tag for a wide variety of protein research.
title_full	Engineering a novel multifunctional green fluorescent protein tag for a wide variety of protein research.
title_fullStr	Engineering a novel multifunctional green fluorescent protein tag for a wide variety of protein research.
title_full_unstemmed	Engineering a novel multifunctional green fluorescent protein tag for a wide variety of protein research.
title_sort	engineering a novel multifunctional green fluorescent protein tag for a wide variety of protein research.
publisher	Public Library of Science (PLoS)
publishDate	2008
url	https://doaj.org/article/2749fe4599ca462299c566b4b19ff582
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Engineering a novel multifunctional green fluorescent protein tag for a wide variety of protein research.

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