Two-photon directed evolution of green fluorescent proteins

Directed evolution has been used extensively to improve the properties of a variety of fluorescent proteins (FPs). Evolutionary strategies, however, have not yet been used to improve the two-photon absorption (2PA) properties of a fluorescent protein, properties that are important for two-photon ima...

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Autores principales: Stoltzfus, Caleb R., Barnett, Lauren M., Drobizhev, Mikhail, Wicks, Geoffrey, Mikhaylov, Alexander, Hughes, Thomas E., Rebane, Aleksander
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2015
Materias:
Article
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4491718/
https://www.ncbi.nlm.nih.gov/pubmed/26145791
http://dx.doi.org/10.1038/srep11968
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author Stoltzfus, Caleb R.
Barnett, Lauren M.
Drobizhev, Mikhail
Wicks, Geoffrey
Mikhaylov, Alexander
Hughes, Thomas E.
Rebane, Aleksander
author_facet Stoltzfus, Caleb R.
Barnett, Lauren M.
Drobizhev, Mikhail
Wicks, Geoffrey
Mikhaylov, Alexander
Hughes, Thomas E.
Rebane, Aleksander
author_sort Stoltzfus, Caleb R.
collection PubMed
description Directed evolution has been used extensively to improve the properties of a variety of fluorescent proteins (FPs). Evolutionary strategies, however, have not yet been used to improve the two-photon absorption (2PA) properties of a fluorescent protein, properties that are important for two-photon imaging in living tissues, including the brain. Here we demonstrate a technique for quantitatively screening the two-photon excited fluorescence (2PEF) efficiency and 2PA cross section of tens of thousands of mutant FPs expressed in E. coli colonies. We use this procedure to move EGFP through three rounds of two-photon directed evolution leading to new variants showing up to a 50% enhancement in peak 2PA cross section and brightness within the near-IR tissue transparency wavelength range.
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spelling pubmed-44917182015-07-08 Two-photon directed evolution of green fluorescent proteins Stoltzfus, Caleb R. Barnett, Lauren M. Drobizhev, Mikhail Wicks, Geoffrey Mikhaylov, Alexander Hughes, Thomas E. Rebane, Aleksander Sci Rep Article Directed evolution has been used extensively to improve the properties of a variety of fluorescent proteins (FPs). Evolutionary strategies, however, have not yet been used to improve the two-photon absorption (2PA) properties of a fluorescent protein, properties that are important for two-photon imaging in living tissues, including the brain. Here we demonstrate a technique for quantitatively screening the two-photon excited fluorescence (2PEF) efficiency and 2PA cross section of tens of thousands of mutant FPs expressed in E. coli colonies. We use this procedure to move EGFP through three rounds of two-photon directed evolution leading to new variants showing up to a 50% enhancement in peak 2PA cross section and brightness within the near-IR tissue transparency wavelength range. Nature Publishing Group 2015-07-06 /pmc/articles/PMC4491718/ /pubmed/26145791 http://dx.doi.org/10.1038/srep11968 Text en Copyright © 2015, Macmillan Publishers Limited 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
Stoltzfus, Caleb R.
Barnett, Lauren M.
Drobizhev, Mikhail
Wicks, Geoffrey
Mikhaylov, Alexander
Hughes, Thomas E.
Rebane, Aleksander
Two-photon directed evolution of green fluorescent proteins
title Two-photon directed evolution of green fluorescent proteins
title_full Two-photon directed evolution of green fluorescent proteins
title_fullStr Two-photon directed evolution of green fluorescent proteins
title_full_unstemmed Two-photon directed evolution of green fluorescent proteins
title_short Two-photon directed evolution of green fluorescent proteins
title_sort two-photon directed evolution of green fluorescent proteins
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4491718/
https://www.ncbi.nlm.nih.gov/pubmed/26145791
http://dx.doi.org/10.1038/srep11968
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