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ID 60635
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Author
Mori, Yoshihiro Graduate School of Natural Science and Technology, Okayama University
Yoshida, Yuki Sony Computer Science Laboratories
Satoh, Ayano Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University ORCID Kaken ID publons researchmap
Moriya, Hisao Research Core for Interdisciplinary Sciences, Okayama University ORCID Kaken ID publons researchmap
Abstract
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 similar to 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.
Keywords
Biological techniques
Cell biology
Gene expression analysis
Molecular biology
Protein translocation
Protein transport
Published Date
2020-03-16
Publication Title
Scientific Reports
Volume
volume10
Issue
issue1
Publisher
Nature
Start Page
4798
ISSN
2045-2322
Content Type
Journal Article
language
英語
OAI-PMH Set
岡山大学
Copyright Holders
© The Author(s) 2020
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publisher
PubMed ID
DOI
Web of Science KeyUT
Related Url
isVersionOf https://doi.org/10.1038/s41598-020-61646-3
License
http://creativecommons.org/licenses/by/4.0/
Funder Name
Japan Society for the Promotion of Science
助成番号
18K19300
17H03618
18K06133