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Development of an experimental method of systematically estimating protein expression limits in HEK293 cells

Protein overexpression sometimes causes cellular defects, although the underlying mechanism is still unknown. A protein’s expression limit, which triggers cellular defects, is a useful indication of the underlying mechanism. In this study, we developed an experimental method of estimating the expres...

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Autores principales: Mori, Yoshihiro, Yoshida, Yuki, Satoh, Ayano, Moriya, Hisao
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7075890/
https://www.ncbi.nlm.nih.gov/pubmed/32179769
http://dx.doi.org/10.1038/s41598-020-61646-3
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author Mori, Yoshihiro
Yoshida, Yuki
Satoh, Ayano
Moriya, Hisao
author_facet Mori, Yoshihiro
Yoshida, Yuki
Satoh, Ayano
Moriya, Hisao
author_sort Mori, Yoshihiro
collection PubMed
description Protein overexpression sometimes causes cellular defects, although the underlying mechanism is still unknown. A protein’s expression limit, which triggers cellular defects, is a useful indication of the underlying mechanism. In this study, we developed an experimental method of estimating the expression limits of target proteins in the human embryonic kidney cell line HEK293 by measuring the proteins’ expression levels in cells that survived after the high-copy introduction of plasmid DNA by which the proteins were expressed under a strong cytomegalovirus promoter. The expression limits of nonfluorescent target proteins were indirectly estimated by measuring the levels of green fluorescent protein (GFP) connected to the target proteins with the self-cleaving sequence P2A. The expression limit of a model GFP was ~5.0% of the total protein, and sustained GFP overexpression caused cell death. The expression limits of GFPs with mitochondria-targeting signals and endoplasmic reticulum localization signals were 1.6% and 0.38%, respectively. The expression limits of four proteins involved in vesicular trafficking were far lower compared to a red fluorescent protein. The protein expression limit estimation method developed will be valuable for defining toxic proteins and consequences of protein overexpression.
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spelling pubmed-70758902020-03-22 Development of an experimental method of systematically estimating protein expression limits in HEK293 cells Mori, Yoshihiro Yoshida, Yuki Satoh, Ayano Moriya, Hisao Sci Rep Article Protein overexpression sometimes causes cellular defects, although the underlying mechanism is still unknown. A protein’s expression limit, which triggers cellular defects, is a useful indication of the underlying mechanism. In this study, we developed an experimental method of estimating the expression limits of target proteins in the human embryonic kidney cell line HEK293 by measuring the proteins’ expression levels in cells that survived after the high-copy introduction of plasmid DNA by which the proteins were expressed under a strong cytomegalovirus promoter. The expression limits of nonfluorescent target proteins were indirectly estimated by measuring the levels of green fluorescent protein (GFP) connected to the target proteins with the self-cleaving sequence P2A. The expression limit of a model GFP was ~5.0% of the total protein, and sustained GFP overexpression caused cell death. The expression limits of GFPs with mitochondria-targeting signals and endoplasmic reticulum localization signals were 1.6% and 0.38%, respectively. The expression limits of four proteins involved in vesicular trafficking were far lower compared to a red fluorescent protein. The protein expression limit estimation method developed will be valuable for defining toxic proteins and consequences of protein overexpression. Nature Publishing Group UK 2020-03-16 /pmc/articles/PMC7075890/ /pubmed/32179769 http://dx.doi.org/10.1038/s41598-020-61646-3 Text en © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Mori, Yoshihiro
Yoshida, Yuki
Satoh, Ayano
Moriya, Hisao
Development of an experimental method of systematically estimating protein expression limits in HEK293 cells
title Development of an experimental method of systematically estimating protein expression limits in HEK293 cells
title_full Development of an experimental method of systematically estimating protein expression limits in HEK293 cells
title_fullStr Development of an experimental method of systematically estimating protein expression limits in HEK293 cells
title_full_unstemmed Development of an experimental method of systematically estimating protein expression limits in HEK293 cells
title_short Development of an experimental method of systematically estimating protein expression limits in HEK293 cells
title_sort development of an experimental method of systematically estimating protein expression limits in hek293 cells
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7075890/
https://www.ncbi.nlm.nih.gov/pubmed/32179769
http://dx.doi.org/10.1038/s41598-020-61646-3
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