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

Yoshihiro Mori,Hisao Moriya,Ayano Satoh,Yuki Yoshida

doi:10.1038/s41598-020-61646-3

Yoshihiro Mori, Hisao Moriya + Show 2 more

Open Access

https://doi.org/10.1038/s41598-020-61646-3

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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.

Highlights

Protein overexpression sometimes causes cellular defects, the underlying mechanism is still unknown
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)
To develop an experimental method of measuring a target protein’s expression limit, we used a green fluorescent protein (GFP) as a target protein because its expression level is estimated by fluorescence of the cells expressing it

Summary

Introduction

Protein overexpression sometimes causes cellular defects, the underlying mechanism is still unknown. 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 underlying mechanism causing these defects is still unknown, it can be estimated using the protein expression limit, which triggers cellular defects[3,4]. The expression limits obtained were useful for classifying the mechanisms underlying cellular defects triggered by protein overexpression[4,11]. 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). 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)

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