Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein

Abstract

Fluorescent proteins are genetically encoded, easily imaged reporters crucial in biology and biotechnology. When a protein is tagged by fusion to a fluorescent protein, interactions between fluorescent proteins can undesirably disturb targeting or function. Unfortunately, all wild-type yellow-to-red fluorescent proteins reported so far are obligately tetrameric and often toxic or disruptive. The first true monomer was mRFP1, derived from the Discosoma sp. fluorescent protein "DsRed" by directed evolution first to increase the speed of maturation, then to break each subunit interface while restoring fluorescence, which cumulatively required 33 substitutions. Although mRFP1 has already proven widely useful, several properties could bear improvement and more colors would be welcome. We report the next generation of monomers. The latest red version matures more completely, is more tolerant of N-terminal fusions and is over tenfold more photostable than mRFP1. Three monomers with distinguishable hues from yellow-orange to red-orange have higher quantum efficiencies.

Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs

“…The application of the GFP technology for imaging of intracellular proteins has been comprehensively reviewed by Tsien, the 2008 Nobel laureate (Tsien 1998). Currently a whole palette of fluorescent proteins, also from coral species, emitting from violet to red are applied extensively as genetically encoded, brightly fluorescent markers in cell biology (Zhang et al 2002;Miyawaki et al 2003;Shaner et al 2004Shaner et al , 2005Verkhusha and Lukyanov 2004;Giepmans et al 2006). Thus far, the enhanced forms of cyan fluorescent protein (ECFP) and yellow fluorescent protein (EYFP) are the most commonly used FRET pair in cell biology.…”

A novel imaging‐based high‐throughput screening approach to anti‐angiogenic drug discovery

“…GFP was expressed from pCGFP (15). tdTomato was expressed from pCtdTomato, a derivative of pCGFP with the GFP replaced by tdTomato (16).…”

An improved mRFP1 adds red to bimolecular fluorescence complementation

“…During the course of this study reports of improved mRFP1 variants were published 8 . Notably, some of these mutants also carried the fluorophore mutation Q66T, but always in concert with additional mutations.…”

“…Our in vitro and in vivo data demonstrate that mRFP1-Q66T represents an improved, useful and readily detectable reporter protein with fluorescence levels comparable to that of the brightest monomeric DsRED variants presently available. mRFP1-Q66T represents a good compromise between enhanced brightness and fast maturation because the additional mutations present in mOrange 8 , while increasing the brightness twofold, seem to negatively affect the maturation time ( Table 1).…”