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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...

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Autores principales: Shibata, Akihiro C. E., Maebashi, Hiroshi K., Nakahata, Yoshihisa, Nabekura, Junichi, Murakoshi, Hideji
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2015
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.
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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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