Quantitative co-expression of proteins at the single cell level--application to a multimeric FRET sensor.
BACKGROUND: Co-expression of proteins is generally achieved by introducing two (or more) independent plasmids into cells, each driving the expression of a different protein of interest. However, the relative expression levels may vary strongly between individual cells and cannot be controlled. Ideal...
Main Authors: | , , , , , |
---|---|
Format: | Article |
Language: | English |
Published: |
Public Library of Science (PLoS)
2011-01-01
|
Series: | PLoS ONE |
Online Access: | http://europepmc.org/articles/PMC3219669?pdf=render |
_version_ | 1811212707740778496 |
---|---|
author | Joachim Goedhart Laura van Weeren Merel J W Adjobo-Hermans Ies Elzenaar Mark A Hink Theodorus W J Gadella |
author_facet | Joachim Goedhart Laura van Weeren Merel J W Adjobo-Hermans Ies Elzenaar Mark A Hink Theodorus W J Gadella |
author_sort | Joachim Goedhart |
collection | DOAJ |
description | BACKGROUND: Co-expression of proteins is generally achieved by introducing two (or more) independent plasmids into cells, each driving the expression of a different protein of interest. However, the relative expression levels may vary strongly between individual cells and cannot be controlled. Ideally, co-expression occurs at a defined ratio, which is constant among cells. This feature is of particular importance for quantitative single cell studies, especially those employing bimolecular Förster Resonance Energy Transfer (FRET) sensors. METHODOLOGY/PRINCIPAL FINDINGS: Four co-expression strategies based on co-transfection, a dual promotor plasmid, an internal ribosome entry site (IRES) and a viral 2A peptide were selected. Co-expression of two spectrally separable fluorescent proteins in single living cells was quantified. It is demonstrated that the 2A peptide strategy can be used for robust equimolar co-expression, while the IRES sequence allows expression of two proteins at a ratio of approximately 3:1. Combined 2A and IRES elements were used for the construction of a single plasmid that drives expression of three individual proteins, which generates a FRET sensor for measuring heterotrimeric G-protein activation. The plasmid drives co-expression of donor and acceptor tagged subunits, with reduced heterogeneity, and can be used to measure G-protein activation in single living cells. CONCLUSIONS/SIGNIFICANCE: Quantitative co-expression of two or more proteins can be achieved with little cell-to-cell variability. This finding enables reliable co-expression of donor and acceptor tagged proteins for FRET studies, which is of particular importance for the development of novel bimolecular sensors that can be expressed from single plasmid. |
first_indexed | 2024-04-12T05:33:36Z |
format | Article |
id | doaj.art-b4af9cd9a1fb4c00a7d31d143907ddba |
institution | Directory Open Access Journal |
issn | 1932-6203 |
language | English |
last_indexed | 2024-04-12T05:33:36Z |
publishDate | 2011-01-01 |
publisher | Public Library of Science (PLoS) |
record_format | Article |
series | PLoS ONE |
spelling | doaj.art-b4af9cd9a1fb4c00a7d31d143907ddba2022-12-22T03:45:56ZengPublic Library of Science (PLoS)PLoS ONE1932-62032011-01-01611e2732110.1371/journal.pone.0027321Quantitative co-expression of proteins at the single cell level--application to a multimeric FRET sensor.Joachim GoedhartLaura van WeerenMerel J W Adjobo-HermansIes ElzenaarMark A HinkTheodorus W J GadellaBACKGROUND: Co-expression of proteins is generally achieved by introducing two (or more) independent plasmids into cells, each driving the expression of a different protein of interest. However, the relative expression levels may vary strongly between individual cells and cannot be controlled. Ideally, co-expression occurs at a defined ratio, which is constant among cells. This feature is of particular importance for quantitative single cell studies, especially those employing bimolecular Förster Resonance Energy Transfer (FRET) sensors. METHODOLOGY/PRINCIPAL FINDINGS: Four co-expression strategies based on co-transfection, a dual promotor plasmid, an internal ribosome entry site (IRES) and a viral 2A peptide were selected. Co-expression of two spectrally separable fluorescent proteins in single living cells was quantified. It is demonstrated that the 2A peptide strategy can be used for robust equimolar co-expression, while the IRES sequence allows expression of two proteins at a ratio of approximately 3:1. Combined 2A and IRES elements were used for the construction of a single plasmid that drives expression of three individual proteins, which generates a FRET sensor for measuring heterotrimeric G-protein activation. The plasmid drives co-expression of donor and acceptor tagged subunits, with reduced heterogeneity, and can be used to measure G-protein activation in single living cells. CONCLUSIONS/SIGNIFICANCE: Quantitative co-expression of two or more proteins can be achieved with little cell-to-cell variability. This finding enables reliable co-expression of donor and acceptor tagged proteins for FRET studies, which is of particular importance for the development of novel bimolecular sensors that can be expressed from single plasmid.http://europepmc.org/articles/PMC3219669?pdf=render |
spellingShingle | Joachim Goedhart Laura van Weeren Merel J W Adjobo-Hermans Ies Elzenaar Mark A Hink Theodorus W J Gadella Quantitative co-expression of proteins at the single cell level--application to a multimeric FRET sensor. PLoS ONE |
title | Quantitative co-expression of proteins at the single cell level--application to a multimeric FRET sensor. |
title_full | Quantitative co-expression of proteins at the single cell level--application to a multimeric FRET sensor. |
title_fullStr | Quantitative co-expression of proteins at the single cell level--application to a multimeric FRET sensor. |
title_full_unstemmed | Quantitative co-expression of proteins at the single cell level--application to a multimeric FRET sensor. |
title_short | Quantitative co-expression of proteins at the single cell level--application to a multimeric FRET sensor. |
title_sort | quantitative co expression of proteins at the single cell level application to a multimeric fret sensor |
url | http://europepmc.org/articles/PMC3219669?pdf=render |
work_keys_str_mv | AT joachimgoedhart quantitativecoexpressionofproteinsatthesinglecelllevelapplicationtoamultimericfretsensor AT lauravanweeren quantitativecoexpressionofproteinsatthesinglecelllevelapplicationtoamultimericfretsensor AT mereljwadjobohermans quantitativecoexpressionofproteinsatthesinglecelllevelapplicationtoamultimericfretsensor AT ieselzenaar quantitativecoexpressionofproteinsatthesinglecelllevelapplicationtoamultimericfretsensor AT markahink quantitativecoexpressionofproteinsatthesinglecelllevelapplicationtoamultimericfretsensor AT theodoruswjgadella quantitativecoexpressionofproteinsatthesinglecelllevelapplicationtoamultimericfretsensor |