title: Quantitative co-expression of proteins at the single cell level--application to a multimeric FRET sensor.
abstract: 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.
  
  
  RESULTS
  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
  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.
authors: Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands. j.goedhart@uva.nl
authors: Joachim
authors: J
authors: Goedhart
authors: Laura
authors: L
authors: van Weeren
authors: Merel J W
authors: MJ
authors: Adjobo-Hermans
authors: Ies
authors: I
authors: Elzenaar
authors: Mark A
authors: MA
authors: Hink
authors: Theodorus W J
authors: TW
authors: Gadella
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D031541:Fluorescence Resonance Energy Transfer; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D043562:Receptors, G-Protein-Coupled / Q000032:analysis*; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; D010446:Peptide Fragments; D043562:Receptors, G-Protein-Coupled; D014764:Viral Proteins; C485184:enhanced green fluorescent protein; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins