| RRC ID |
69148
|
| Author |
Mori Y, Yoshida Y, Satoh A, Moriya H.
|
| Title |
Development of an experimental method of systematically estimating protein expression limits in HEK293 cells.
|
| Journal |
Sci Rep
|
| 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 ~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.
|
| Volume |
10(1)
|
| Pages |
4798
|
| Published |
2020-3-16
|
| DOI |
10.1038/s41598-020-61646-3
|
| PII |
10.1038/s41598-020-61646-3
|
| PMID |
32179769
|
| PMC |
PMC7075890
|
| MeSH |
Cell Death / genetics*
Cytomegalovirus / genetics
DNA
Endoplasmic Reticulum
Gene Expression*
Green Fluorescent Proteins / genetics
Green Fluorescent Proteins / metabolism*
HEK293 Cells
Humans
Luminescent Proteins / genetics
Luminescent Proteins / metabolism
Methotrexate
Plasmids
Promoter Regions, Genetic
Protein Biosynthesis / genetics*
Protein Transport
Tetrahydrofolate Dehydrogenase
|
| IF |
3.998
|
| Resource |
| DNA material |
pCMV-GFP (RDB19277)
pCMV-MTS-GFP (RDB19278)
pCMV-ER-GFP (RDB19279)
pCMV-RFP (RDB19280)
pCMV-RFP* (RDB19281)
pCMV-SEAP (RDB19282)
pCMV-HAS (RDB19283)
pCMV-SEAPΔSS (RDB19284)
pCMV-SEC24 (RDB19285)
pCMV-SEC31 (RDB19286)
pCMV-RAB1 (RDB19287)
pCMV-RAB5 (RDB19288)
pCMV-GFP-D (RDB19289) |
