In vivo characterisation of fluorescent proteins in budding yeast

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.

A yeast FRET biosensor enlightens cAMP signalling

“…It shows an exceptionally high a FRET range, up to a normalized ratio change of 1.8. Moreover the usage of mTq2-tdTomato as a FRET pair results in decreased pH-sensitivity and increased photostability of the sensor, compared to YFP-CFP based sensors 55 . Signalling mutants and the non-responsive yEPAC variant showed good cAMP selectivity of the sensor.…”

“…The FRET-pairs mCherry-T2A-mTurquoise2 (mTq2), tagRFP-T2A-mTq2, tagRFPT-T2A-mTq2 and tdTomato-T2A-mTq2 in pDRF1-GW were previously constructed 55 . mCherry-mTq2, tagRFP-mTq2 and tagRFPT-mTq2 in a clontech-style C1 mammalian expression vector were obtained from Mastop et al 56 , digested using NheI and NotI (New England Biolabs, Ipswich, Massachusetts, USA), and ligated with T4 ligase (New England Biolabs) in the yeast expression vector pDRF1-GW 55 , digested with the same restriction enzymes. mTq2 in pDRF1-GW was created by performing a PCR, using KOD polymerase (Merck-Millipore, Burlington, Massachusetts, USA) on mTq2-C1 using forward primer 5'-AGGTCTATATAAGCAGAGC-3' and reverse primer 5'-TAGCGGCCGCTTACTTGTACAGCTCGTCCATG-3'.…”

“…In yeast, cAMP signalling is believed to be partly initiated by intracellular acidification, which occurs transiently when derepressed cells start to metabolize fermentable sugars 6,10 . Since Venus is pH sensitive 55,60 , we opted to replace it for a pH-robust acceptor fluorescent protein (FP). We tested mCherry, tagRFP, tagRFP-T and tdTomato as potential acceptors, as we have previously shown that these proteins are more pH-insensitive 55 .…”

“…Since Venus is pH sensitive 55,60 , we opted to replace it for a pH-robust acceptor fluorescent protein (FP). We tested mCherry, tagRFP, tagRFP-T and tdTomato as potential acceptors, as we have previously shown that these proteins are more pH-insensitive 55 . We replaced the Venus acceptor for tdTomato as this was the best acceptor for mTurquoise2; It has a FRET efficiency of 23% and a substantial sensitized emission ( Fig.…”

“…We also tested the maximal temporal resolution we could obtain. The mTq2-tdTomato FRET pair is highly photostable 55 , making the FRET sensor useful for both extended (over many hours) and high temporal resolution (rapid frame rate) time-lapse movies. Using yEPAC, we could record cAMP responses up to 15 minutes with a 3 second time interval, i.e.…”