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Development of a Molecularly Evolved, Highly Sensitive CaMKII FRET Sensor with Improved Expression Pattern
Genetically encoded fluorescence resonance energy transfer (FRET) biosensors have been successfully used to visualize protein activity in living cells. The sensitivity and accuracy of FRET measurements directly depend on biosensor folding efficiency, expression pattern, sensitivity, and dynamic rang...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4370617/ https://www.ncbi.nlm.nih.gov/pubmed/25799407 http://dx.doi.org/10.1371/journal.pone.0121109 |
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author | Shibata, Akihiro C. E. Maebashi, Hiroshi K. Nakahata, Yoshihisa Nabekura, Junichi Murakoshi, Hideji |
author_facet | Shibata, Akihiro C. E. Maebashi, Hiroshi K. Nakahata, Yoshihisa Nabekura, Junichi Murakoshi, Hideji |
author_sort | Shibata, Akihiro C. E. |
collection | PubMed |
description | Genetically encoded fluorescence resonance energy transfer (FRET) biosensors have been successfully used to visualize protein activity in living cells. The sensitivity and accuracy of FRET measurements directly depend on biosensor folding efficiency, expression pattern, sensitivity, and dynamic range. Here, to improve the folding efficiency of the Ca(2+)/calmodulin-dependent protein kinase II alpha (CaMKIIα) FRET biosensor, we amplified the association domain of the CaMKIIα gene using error-prone polymerase chain reaction (PCR) and fused it to the N-terminus of mCherry in a bacterial expression vector. We also created an Escherichia coli expression library based on a previously reported fluorescent protein folding reporter method, and found a bright red fluorescent colony that contained the association domain with four mutations (F394L, I419V, A430T, and I434T). In vitro assays using the purified mutant protein confirmed improved folding kinetics of the downstream fluorescent protein, but not of the association domain itself. Furthermore, we introduced these mutations into the previously reported CaMKIIα FRET sensor and monitored its Ca(2+)/calmodulin-dependent activation in HeLa cells using 2-photon fluorescence lifetime imaging microscopy (2pFLIM), and found that the expression pattern and signal reproducibility of the mutant sensor were greatly improved without affecting the autophosphorylation function and incorporation into oligomeric CaMKIIα. We believe that our improved CaMKIIα FRET sensor would be useful in various types of cells and tissues, providing data with high accuracy and reproducibility. In addition, the method described here may also be applicable for improving the performance of all currently available FRET sensors. |
format | Online Article Text |
id | pubmed-4370617 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-43706172015-04-04 Development of a Molecularly Evolved, Highly Sensitive CaMKII FRET Sensor with Improved Expression Pattern Shibata, Akihiro C. E. Maebashi, Hiroshi K. Nakahata, Yoshihisa Nabekura, Junichi Murakoshi, Hideji PLoS One Research Article Genetically encoded fluorescence resonance energy transfer (FRET) biosensors have been successfully used to visualize protein activity in living cells. The sensitivity and accuracy of FRET measurements directly depend on biosensor folding efficiency, expression pattern, sensitivity, and dynamic range. Here, to improve the folding efficiency of the Ca(2+)/calmodulin-dependent protein kinase II alpha (CaMKIIα) FRET biosensor, we amplified the association domain of the CaMKIIα gene using error-prone polymerase chain reaction (PCR) and fused it to the N-terminus of mCherry in a bacterial expression vector. We also created an Escherichia coli expression library based on a previously reported fluorescent protein folding reporter method, and found a bright red fluorescent colony that contained the association domain with four mutations (F394L, I419V, A430T, and I434T). In vitro assays using the purified mutant protein confirmed improved folding kinetics of the downstream fluorescent protein, but not of the association domain itself. Furthermore, we introduced these mutations into the previously reported CaMKIIα FRET sensor and monitored its Ca(2+)/calmodulin-dependent activation in HeLa cells using 2-photon fluorescence lifetime imaging microscopy (2pFLIM), and found that the expression pattern and signal reproducibility of the mutant sensor were greatly improved without affecting the autophosphorylation function and incorporation into oligomeric CaMKIIα. We believe that our improved CaMKIIα FRET sensor would be useful in various types of cells and tissues, providing data with high accuracy and reproducibility. In addition, the method described here may also be applicable for improving the performance of all currently available FRET sensors. Public Library of Science 2015-03-23 /pmc/articles/PMC4370617/ /pubmed/25799407 http://dx.doi.org/10.1371/journal.pone.0121109 Text en © 2015 Shibata et al http://creativecommons.org/licenses/by/4.0/ 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 properly credited. |
spellingShingle | Research Article Shibata, Akihiro C. E. Maebashi, Hiroshi K. Nakahata, Yoshihisa Nabekura, Junichi Murakoshi, Hideji Development of a Molecularly Evolved, Highly Sensitive CaMKII FRET Sensor with Improved Expression Pattern |
title | Development of a Molecularly Evolved, Highly Sensitive CaMKII FRET Sensor with Improved Expression Pattern |
title_full | Development of a Molecularly Evolved, Highly Sensitive CaMKII FRET Sensor with Improved Expression Pattern |
title_fullStr | Development of a Molecularly Evolved, Highly Sensitive CaMKII FRET Sensor with Improved Expression Pattern |
title_full_unstemmed | Development of a Molecularly Evolved, Highly Sensitive CaMKII FRET Sensor with Improved Expression Pattern |
title_short | Development of a Molecularly Evolved, Highly Sensitive CaMKII FRET Sensor with Improved Expression Pattern |
title_sort | development of a molecularly evolved, highly sensitive camkii fret sensor with improved expression pattern |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4370617/ https://www.ncbi.nlm.nih.gov/pubmed/25799407 http://dx.doi.org/10.1371/journal.pone.0121109 |
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