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Quantitative Co-Expression of Proteins at the Single Cell Level – Application to a Multimeric FRET Sensor 1 1 1,2 1 1 Joachim Goedhart *, Laura van Weeren , Merel J.W. Adjobo-Hermans , Ies Elzenaar , Mark A. Hink , Theodorus W.J. Gadella, Jr.1* 1 Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands, 2 Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Radboud University, Nijmegen, The Netherlands 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 ¨ Forster 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. Co