The basic principle of this analysis is that we can assume that transfection creates a cell population harboring diverse plasmid copy numbers, leading to diverse expression levels of a target protein encoded on the plasmid, because the process is random. Therefore, if an experimental condition is established that can estimate the expression limit of GFP, it should be applicable to the majority of other proteins with lower expression limits compared to GFP.įigure 1A shows the experimental procedure. GFP is considered a nonharmful protein 13, 14, 15, and its expression limit in yeast is ~15% of the total protein, which is highest among other proteins in yeast 5, 7. We used moxGFP, which folds quickly and does not misfold in the endoplasmic reticulum (ER) 12. To develop an experimental method of measuring a target protein’s expression limit, we used a GFP as a target protein because its expression level is easily estimated by fluorescence of the cells expressing it. Estimation of expression limits slightly but significantly improved by concentrating cells with higher expression levels of the target protein using the antagonism between dihydrofolate reductase (DHFR) and its inhibitor methotrexate (MTX).Įstimation of GFP expression limit with high-efficient transfection We also indirectly estimated the expression limits of nonfluorescent target proteins by measuring the levels of GFP connected to the target proteins with the self-cleaving sequence P2A by measuring GFP fluorescence.
In this study, we developed an experimental method of systematically evaluating the expression limits of target proteins in the human embryonic kidney cell line HEK293 by measuring the target protein level in cells surviving after high-efficient, multicopy introduction of plasmid DNA by which the target protein is highly expressed under a cytomegalovirus promoter (CMV-pro).įor proof-of-concept, we estimated the expression limits of a green fluorescent protein (GFP) and its derivatives as model proteins. The expression limits obtained were useful for classifying the mechanisms underlying cellular defects triggered by protein overexpression 4, 11. We previously developed an experimental genetic tug-of-war (gTOW) method of estimating the expression limits of target proteins in yeasts 8, 9, 10. Transported proteins should have lower expression limits than cytoplasmic proteins because the overexpression of transported proteins overloads resources for protein transport 5, 7. For example, a protein with the highest expression limit is considered harmless, and its ultimate overexpression causes overloading of protein synthesis 5, 6. Although the underlying mechanism causing these defects is still unknown, it can be estimated using the protein expression limit, which triggers cellular defects 3, 4. Protein overexpression sometimes causes cellular defects 1, 2. The protein expression limit estimation method developed will be valuable for defining toxic proteins and consequences of protein overexpression.
The expression limits of four proteins involved in vesicular trafficking were far lower compared to a red fluorescent protein. The expression limits of GFPs with mitochondria-targeting signals and endoplasmic reticulum localization signals were 1.6% and 0.38%, respectively. 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 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.
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. A protein’s expression limit, which triggers cellular defects, is a useful indication of the underlying mechanism. Protein overexpression sometimes causes cellular defects, although the underlying mechanism is still unknown.
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