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Updated: May 27, 2026
A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts
Published on: December 1, 2018
Quantitative co-expression of proteins at the single cell level--application to a multimeric FRET sensor
Joachim Goedhart1, Laura van Weeren, Merel J W Adjobo-Hermans
1Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands. j.goedhart@uva.nl
Achieve precise protein co-expression using viral 2A peptides or internal ribosome entry site (IRES) elements for consistent single-cell studies. This enables robust development of Förster Resonance Energy Transfer (FRET) sensors and quantitative biological measurements.
Area of Science:
- Molecular Biology
- Biotechnology
- Cell Biology
Background:
- Traditional protein co-expression methods using multiple plasmids lead to variable expression levels across cells.
- Controlled, consistent co-expression ratios are crucial for quantitative single-cell analyses, particularly for Förster Resonance Energy Transfer (FRET) studies.
Purpose of the Study:
- To evaluate and compare different strategies for achieving controlled protein co-expression in single cells.
- To develop a single-plasmid system for reliable co-expression of multiple proteins for advanced biological sensing.
Main Methods:
- Compared co-transfection, dual promoter plasmids, internal ribosome entry site (IRES), and viral 2A peptides for co-expressing fluorescent proteins.
- Quantified co-expression levels and cell-to-cell variability using these strategies.
- Constructed and tested a single plasmid utilizing combined 2A and IRES elements for expressing three proteins.
Main Results:
- Viral 2A peptide strategy enables robust equimolar co-expression.
- IRES sequences facilitate protein expression at an approximate 3:1 ratio.
- A single plasmid combining 2A and IRES elements successfully drove co-expression of three proteins, generating a functional FRET sensor for G-protein activation with reduced heterogeneity.
Conclusions:
- Quantitative co-expression with minimal cell-to-cell variability is achievable.
- This facilitates reliable co-expression of tagged proteins for FRET studies.
- Enables the development of novel bimolecular sensors expressed from a single plasmid for precise biological measurements.
