mFruit variant mCherry2 is a further engineered variant of mCherry that retains similar excitation and emission maxima (λex = 589 nm and λem = 610 nm) but has slightly higher brightness. This variant was generated some years ago by several rounds of directed evolution of an mCherry-ferritin fusion, as was originally described for Superfolder GFP (Waldo et al, 1999), but was not characterized in detail until the current work
The mCherry-K92N/K138C/K139R/N196D variant, with just four of the six mCherry2 mutations, permitted a relative faster E. coli growth rate than EGFP, suggesting that this variant (designated as mCherry1.5) had even lower cytotoxicity than EGFP and mCherry2... Unfortunately, both mCherry1.5 and mCherry itself showed similar patterns of protein mislocalization in these fusions, as compared to identical mEGFP fusions, indicating that the low bacterial cytotoxicity of an RFP does not necessarily correlate with more faithful fusion protein localization. Based on this result and our cumulative experience with engineering RFPs, we suggest that ameliorating the mislocalization of RFPs may ultimately require a high-throughput image-based screen of mammalian cells expressing a library of RFP variants in the context of a fusion that tends to mislocalize.
The bacterial cytotoxicity assay demonstrated that RDSmCherry1 has cytotoxicity similar to that of mCherry and LSSmCherry1, indicating that the decreased cytotoxicity of mCherry2 was lost during directed evolution.
... 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. ... 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. 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.
The increased in vitro brightness of mCherry2 does not translate into substantially improved brightness in either bacteria or mammalian cells. However, we noticed that E. coli colonies expressing mCherry2 have a larger size than the ones expressing mCherry, at similar colony densities on LB agar plates. This observation suggested that mCherry2 might have decreased cytotoxicity in E. coli.