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Expression of varied GFPs in Saccharomyces cerevisiae: codon optimization yields stronger than expected expression and fluorescence intensity
Green fluorescent protein (GFP), which was originally isolated from jellyfish, is a widely used tool in biological research, and homologs from other organisms are available. However, researchers must determine which GFP is the most suitable for a specific host. Here, we expressed GFPs from several s...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5080575/ https://www.ncbi.nlm.nih.gov/pubmed/27782154 http://dx.doi.org/10.1038/srep35932 |
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author | Kaishima, Misato Ishii, Jun Matsuno, Toshihide Fukuda, Nobuo Kondo, Akihiko |
author_facet | Kaishima, Misato Ishii, Jun Matsuno, Toshihide Fukuda, Nobuo Kondo, Akihiko |
author_sort | Kaishima, Misato |
collection | PubMed |
description | Green fluorescent protein (GFP), which was originally isolated from jellyfish, is a widely used tool in biological research, and homologs from other organisms are available. However, researchers must determine which GFP is the most suitable for a specific host. Here, we expressed GFPs from several sources in codon-optimized and non-codon-optimized forms in the yeast Saccharomyces cerevisiae, which represents an ideal eukaryotic model. Surprisingly, codon-optimized mWasabi and mNeonGreen, which are typically the brightest GFPs, emitted less green fluorescence than did the other five codon-optimized GFPs tested in S. cerevisiae. Further, commercially available GFPs that have been optimized for mammalian codon usage (e.g., EGFP, AcGFP1 and TagGFP2) unexpectedly exhibited extremely low expression levels in S. cerevisiae. In contrast, codon-optimization of the GFPs for S. cerevisiae markedly increased their expression levels, and the fluorescence intensity of the cells increased by a maximum of 101-fold. Among the tested GFPs, the codon-optimized monomeric mUkG1 from soft coral showed the highest levels of both expression and fluorescence. Finally, the expression of this protein as a fusion-tagged protein successfully improved the reporting system’s ability to sense signal transduction and protein–protein interactions in S. cerevisiae and increased the detection rates of target cells using flow cytometry. |
format | Online Article Text |
id | pubmed-5080575 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Nature Publishing Group |
record_format | MEDLINE/PubMed |
spelling | pubmed-50805752016-10-31 Expression of varied GFPs in Saccharomyces cerevisiae: codon optimization yields stronger than expected expression and fluorescence intensity Kaishima, Misato Ishii, Jun Matsuno, Toshihide Fukuda, Nobuo Kondo, Akihiko Sci Rep Article Green fluorescent protein (GFP), which was originally isolated from jellyfish, is a widely used tool in biological research, and homologs from other organisms are available. However, researchers must determine which GFP is the most suitable for a specific host. Here, we expressed GFPs from several sources in codon-optimized and non-codon-optimized forms in the yeast Saccharomyces cerevisiae, which represents an ideal eukaryotic model. Surprisingly, codon-optimized mWasabi and mNeonGreen, which are typically the brightest GFPs, emitted less green fluorescence than did the other five codon-optimized GFPs tested in S. cerevisiae. Further, commercially available GFPs that have been optimized for mammalian codon usage (e.g., EGFP, AcGFP1 and TagGFP2) unexpectedly exhibited extremely low expression levels in S. cerevisiae. In contrast, codon-optimization of the GFPs for S. cerevisiae markedly increased their expression levels, and the fluorescence intensity of the cells increased by a maximum of 101-fold. Among the tested GFPs, the codon-optimized monomeric mUkG1 from soft coral showed the highest levels of both expression and fluorescence. Finally, the expression of this protein as a fusion-tagged protein successfully improved the reporting system’s ability to sense signal transduction and protein–protein interactions in S. cerevisiae and increased the detection rates of target cells using flow cytometry. Nature Publishing Group 2016-10-26 /pmc/articles/PMC5080575/ /pubmed/27782154 http://dx.doi.org/10.1038/srep35932 Text en Copyright © 2016, The Author(s) http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ |
spellingShingle | Article Kaishima, Misato Ishii, Jun Matsuno, Toshihide Fukuda, Nobuo Kondo, Akihiko Expression of varied GFPs in Saccharomyces cerevisiae: codon optimization yields stronger than expected expression and fluorescence intensity |
title | Expression of varied GFPs in Saccharomyces cerevisiae: codon optimization yields stronger than expected expression and fluorescence intensity |
title_full | Expression of varied GFPs in Saccharomyces cerevisiae: codon optimization yields stronger than expected expression and fluorescence intensity |
title_fullStr | Expression of varied GFPs in Saccharomyces cerevisiae: codon optimization yields stronger than expected expression and fluorescence intensity |
title_full_unstemmed | Expression of varied GFPs in Saccharomyces cerevisiae: codon optimization yields stronger than expected expression and fluorescence intensity |
title_short | Expression of varied GFPs in Saccharomyces cerevisiae: codon optimization yields stronger than expected expression and fluorescence intensity |
title_sort | expression of varied gfps in saccharomyces cerevisiae: codon optimization yields stronger than expected expression and fluorescence intensity |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5080575/ https://www.ncbi.nlm.nih.gov/pubmed/27782154 http://dx.doi.org/10.1038/srep35932 |
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