Abstract
Fluorescent proteins (FPs) are widely used in many organisms, but are commonly characterised in vitro. However, the in vitro properties may poorly reflect in vivo performance. Therefore, we characterised 27 FPs in vivo using Saccharomyces cerevisiae as model organism. We linked the FPs via a T2A peptide to a control FP, producing equimolar expression of the 2 FPs from 1 plasmid. Using this strategy, we characterised the FPs for brightness, photostability, photochromicity and pH-sensitivity, achieving a comprehensive in vivo characterisation. Many FPs showed different in vivo properties compared to existing in vitro data. Additionally, various FPs were photochromic, which affects readouts due to complex bleaching kinetics. Finally, we codon optimized the best performing FPs for optimal expression in yeast, and found that codon-optimization alters FP characteristics. These FPs improve experimental signal readout, opening new experimental possibilities. Our results may guide future studies in yeast that employ fluorescent proteins.
| Original language | English |
|---|---|
| Article number | 2234 |
| Pages (from-to) | 1-14 |
| Number of pages | 14 |
| Journal | Scientific Reports |
| Volume | 9 |
| DOIs | |
| Publication status | Published - 19 Feb 2019 |
Funding
We thank Paula Martínez Román (Erasmus scholarship BSc student at Vrije Universiteit Amsterdam) and Jip Wulffelé (BSc student at Vrije Universiteit Amsterdam) for constructing various FP constructs and assisting with experiments. We thank Rick Nijhout for constructing pFA6a-link-ymNeongreen SpHis5. We thank Laura van Weeren and Marieke Mastop (Molecular Cytology, University of Amsterdam) for constructing the plasmids for co-expressing proteins with the T2A sequence in mammalian cells. This work was supported in part by the NWO VICI grant 865.14.005 and the F10.001.03 BE Basic project under the TKIEB01003 AMBIC program.
| Funders | Funder number |
|---|---|
| Nederlandse Organisatie voor Wetenschappelijk Onderzoek | 865.14.005 |