Cargando…

Development of Cysteine-Free Fluorescent Proteins for the Oxidative Environment

Molecular imaging employing fluorescent proteins has been widely used to highlight specific reactions or processes in various fields of the life sciences. Despite extensive improvements of the fluorescent tag, this technology is still limited in the study of molecular events in the extracellular mil...

Descripción completa

Detalles Bibliográficos
Autores principales: Suzuki, Takahisa, Arai, Seisuke, Takeuchi, Mayumi, Sakurai, Chiye, Ebana, Hideaki, Higashi, Tsunehito, Hashimoto, Hitoshi, Hatsuzawa, Kiyotaka, Wada, Ikuo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3359384/
https://www.ncbi.nlm.nih.gov/pubmed/22649538
http://dx.doi.org/10.1371/journal.pone.0037551
_version_ 1782233875212140544
author Suzuki, Takahisa
Arai, Seisuke
Takeuchi, Mayumi
Sakurai, Chiye
Ebana, Hideaki
Higashi, Tsunehito
Hashimoto, Hitoshi
Hatsuzawa, Kiyotaka
Wada, Ikuo
author_facet Suzuki, Takahisa
Arai, Seisuke
Takeuchi, Mayumi
Sakurai, Chiye
Ebana, Hideaki
Higashi, Tsunehito
Hashimoto, Hitoshi
Hatsuzawa, Kiyotaka
Wada, Ikuo
author_sort Suzuki, Takahisa
collection PubMed
description Molecular imaging employing fluorescent proteins has been widely used to highlight specific reactions or processes in various fields of the life sciences. Despite extensive improvements of the fluorescent tag, this technology is still limited in the study of molecular events in the extracellular milieu. This is partly due to the presence of cysteine in the fluorescent proteins. These proteins almost cotranslationally form disulfide bonded oligomers when expressed in the endoplasmic reticulum (ER). Although single molecule photobleaching analysis showed that these oligomers were not fluorescent, the fluorescent monomer form often showed aberrant behavior in folding and motion, particularly when fused to cysteine-containing cargo. Therefore we investigated whether it was possible to eliminate the cysteine without losing the brightness. By site-saturated mutagenesis, we found that the cysteine residues in fluorescent proteins could be replaced with specific alternatives while still retaining their brightness. cf(cysteine-free)SGFP2 showed significantly reduced restriction of free diffusion in the ER and marked improvement of maturation when fused to the prion protein. We further applied this approach to TagRFP family proteins and found a set of mutations that obtains the same level of brightness as the cysteine-containing proteins. The approach used in this study to generate new cysteine-free fluorescent tags should expand the application of molecular imaging to the extracellular milieu and facilitate its usage in medicine and biotechnology.
format Online
Article
Text
id pubmed-3359384
institution National Center for Biotechnology Information
language English
publishDate 2012
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-33593842012-05-30 Development of Cysteine-Free Fluorescent Proteins for the Oxidative Environment Suzuki, Takahisa Arai, Seisuke Takeuchi, Mayumi Sakurai, Chiye Ebana, Hideaki Higashi, Tsunehito Hashimoto, Hitoshi Hatsuzawa, Kiyotaka Wada, Ikuo PLoS One Research Article Molecular imaging employing fluorescent proteins has been widely used to highlight specific reactions or processes in various fields of the life sciences. Despite extensive improvements of the fluorescent tag, this technology is still limited in the study of molecular events in the extracellular milieu. This is partly due to the presence of cysteine in the fluorescent proteins. These proteins almost cotranslationally form disulfide bonded oligomers when expressed in the endoplasmic reticulum (ER). Although single molecule photobleaching analysis showed that these oligomers were not fluorescent, the fluorescent monomer form often showed aberrant behavior in folding and motion, particularly when fused to cysteine-containing cargo. Therefore we investigated whether it was possible to eliminate the cysteine without losing the brightness. By site-saturated mutagenesis, we found that the cysteine residues in fluorescent proteins could be replaced with specific alternatives while still retaining their brightness. cf(cysteine-free)SGFP2 showed significantly reduced restriction of free diffusion in the ER and marked improvement of maturation when fused to the prion protein. We further applied this approach to TagRFP family proteins and found a set of mutations that obtains the same level of brightness as the cysteine-containing proteins. The approach used in this study to generate new cysteine-free fluorescent tags should expand the application of molecular imaging to the extracellular milieu and facilitate its usage in medicine and biotechnology. Public Library of Science 2012-05-23 /pmc/articles/PMC3359384/ /pubmed/22649538 http://dx.doi.org/10.1371/journal.pone.0037551 Text en Suzuki 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
Suzuki, Takahisa
Arai, Seisuke
Takeuchi, Mayumi
Sakurai, Chiye
Ebana, Hideaki
Higashi, Tsunehito
Hashimoto, Hitoshi
Hatsuzawa, Kiyotaka
Wada, Ikuo
Development of Cysteine-Free Fluorescent Proteins for the Oxidative Environment
title Development of Cysteine-Free Fluorescent Proteins for the Oxidative Environment
title_full Development of Cysteine-Free Fluorescent Proteins for the Oxidative Environment
title_fullStr Development of Cysteine-Free Fluorescent Proteins for the Oxidative Environment
title_full_unstemmed Development of Cysteine-Free Fluorescent Proteins for the Oxidative Environment
title_short Development of Cysteine-Free Fluorescent Proteins for the Oxidative Environment
title_sort development of cysteine-free fluorescent proteins for the oxidative environment
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3359384/
https://www.ncbi.nlm.nih.gov/pubmed/22649538
http://dx.doi.org/10.1371/journal.pone.0037551
work_keys_str_mv AT suzukitakahisa developmentofcysteinefreefluorescentproteinsfortheoxidativeenvironment
AT araiseisuke developmentofcysteinefreefluorescentproteinsfortheoxidativeenvironment
AT takeuchimayumi developmentofcysteinefreefluorescentproteinsfortheoxidativeenvironment
AT sakuraichiye developmentofcysteinefreefluorescentproteinsfortheoxidativeenvironment
AT ebanahideaki developmentofcysteinefreefluorescentproteinsfortheoxidativeenvironment
AT higashitsunehito developmentofcysteinefreefluorescentproteinsfortheoxidativeenvironment
AT hashimotohitoshi developmentofcysteinefreefluorescentproteinsfortheoxidativeenvironment
AT hatsuzawakiyotaka developmentofcysteinefreefluorescentproteinsfortheoxidativeenvironment
AT wadaikuo developmentofcysteinefreefluorescentproteinsfortheoxidativeenvironment