Date Published: February 27, 2017
Publisher: Public Library of Science
Author(s): Yi Shen, Yingche Chen, Jiahui Wu, Nathan C. Shaner, Robert E. Campbell, Kurt I. Anderson.
MCherry, the Discosoma sp. mushroom coral-derived monomeric red fluorescent protein (RFP), is a commonly used genetically encoded fluorophore for live cell fluorescence imaging. We have used a combination of protein design and directed evolution to develop mCherry variants with low cytotoxicity to Escherichia coli and altered excitation and emission profiles. These efforts ultimately led to a long Stokes shift (LSS)-mCherry variant (λex = 460 nm and λem = 610 nm) and a red-shifted (RDS)-mCherry variant (λex = 600 nm and λem = 630 nm). These new RFPs provide insight into the influence of the chromophore environment on mCherry’s fluorescence properties, and may serve as templates for the future development of fluorescent probes for live cell imaging.
The discovery of red fluorescent proteins (RFPs) in non-bioluminescent Anthozoa species  was a breakthrough that rivals the inimitable discovery of Aequorea victoria green fluorescent protein (avGFP) [2,3], and the first examples of recombinant expression of avGFP and its color variants [4–7]. Subsequent protein engineering efforts have produced two predominant lineages of monomeric RFPs derived from naturally tetrameric precursors. One lineage is derived from Discosoma sp. mushroom coral and includes the first monomeric (m) RFP, mRFP1 , and the mRFP1-derived “mFruit” variants mCherry, mOrange , and mApple , among others. mCherry is currently the most widely used mRFP for live cell imaging due to its monomeric structure, high brightness, fast maturation, and good photostability . The second lineage is engineered from the Entacmaea quadricolor sea anemone RFPs eqFP578 and eqFP611  and includes TagRFP , mKate , mKate2 , mRuby , mRuby2 , mRuby3 , and FusionRed . All of the above mentioned RFPs have been widely used by the cell biology research community for imaging of protein dynamics and localization in live cells.
Inspired by the observation of decreased bacterial cytotoxicity for mCherry2 (relative to mCherry), we have used protein design and directed evolution to develop new RFP variants with low cytotoxicity to E. coli and altered fluorescence excitation and emission profiles. However, at this stage of development, none of these variants are improved relative to the current state of the art monomeric RFPs in their respective classes. That is, FusionRed is a monomeric RFP with low cytotoxicity, excellent performance in fusions expressed in mammalian cells, and brightness that is practically identical to mCherry2 . mCardinal, which is derived from Entacmaea quadricolor eqFP578, is 3.3× brighter than RDSmCherry1 and has an emission peak that is 29 nm more red-shifted . hmKeima8.5 has similar brightness to LSSmCherry1 and has been demonstrated to provide particularly high brightness and photostability when expressed in mammalian cells . The recently reported CyOFP1 is 3x brighter than LSSmCherry1 . So, while these new mCherry variants are unlikely to find immediate use in live cell imaging applications, they do provide insight into the influence of the protein structure on mCherry’s fluorescence and cytotoxicity properties and may serve as starting points for future probe development efforts.