Quantitative Co-Expression of Proteins at the Single Cell Level – Application to a Multimeric FRET Sensor

Joachim Goedhart,Laura Van Weeren,Theodorus W.J Gadella,Merel J.W Adjobo-Hermans,Ies Elzenaar,Mark A Hink,Anna Maria Delprato

doi:10.1371/journal.pone.0027321

Abstract

BackgroundCo-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 FindingsFour 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/SignificanceQuantitative 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.

Highlights

Encoded Forster Resonance Energy Transfer (FRET) based biosensors have revealed novel insights in spatial and temporal aspects of protein interactions or conformations in a wide variety of cellular processes [1,2]
We found that internal ribosome entry site (IRES) and viral 2A peptides can be used to co-express proteins at a fixed ratio at the single cell level
It is of note that the CFP fluorescence intensity in this experiment is decreased by FRET, and that cellular autofluorescence contributes to both the CFP and the YFP channel, due to low expression of the G-protein subunits

Summary

Introduction

Encoded Forster Resonance Energy Transfer (FRET) based biosensors have revealed novel insights in spatial and temporal aspects of protein interactions or conformations in a wide variety of cellular processes [1,2]. These sensors often consist of two interacting proteins or a protein and an interacting domain sandwiched between a donor and an acceptor fluorophore. Unimolecular sensors require the two interacting proteins or domains to be physically linked, which is not always possible due to structural constraints or post-translational modifications at the C- or N-terminus[4] In such cases the two interacting proteins, fused to donor and acceptor fluorophores, need to be expressed separately. This feature is of particular importance for quantitative single cell studies, especially those employing bimolecular Forster Resonance Energy Transfer (FRET) sensors

Methods

Results

Conclusion