Abstract
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.
Highlights
The discovery of red fluorescent proteins (RFPs) in non-bioluminescent Anthozoa species [1] was a breakthrough that rivals the inimitable discovery of Aequorea victoria green fluorescent protein [2,3], and the first examples of recombinant expression of avGFP and its color variants [4,5,6,7]
In this report we describe efforts to engineer new long Stokes shift and red-shifted RFPs based on mCherry variants with decreased cytotoxicity
To investigate whether the low bacterial toxicity was correlated with improved performance for mammalian cell imaging, mCherry1.5 was expressed in mammalian cells as a fusion to the cytoplasmic end of an endoplasmic reticulum signal-anchor membrane protein (CytERM) [37] and calcium release-activated calcium channel protein 1 (Orai1), which both tend to mislocalize when fused to mCherry [38]. Both mCherry1.5 and mCherry itself showed similar patterns of protein mislocalization in these fusions, as compared to identical mEGFP fusions (S1 Fig), 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 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
Summary
The discovery of red fluorescent proteins (RFPs) in non-bioluminescent Anthozoa species [1] 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 [4,5,6,7]. Subsequent protein engineering efforts have produced two predominant lineages of monomeric RFPs derived from naturally tetrameric precursors. Mushroom coral and includes the first monomeric (m) RFP, mRFP1 [8], and the mRFP1-derived “mFruit” variants mCherry, mOrange [9], and mApple [10], among others. The second lineage is engineered from the Entacmaea quadricolor sea anemone RFPs eqFP578 and eqFP611 [11] and includes TagRFP [12], mKate [13], mKate2 [14], mRuby [15], mRuby2 [16], mRuby3 [17], and FusionRed [18]. 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. A substantial effort has been invested in modifying the properties of typical RFPs (that is, those with λex ~ 570–590 nm and λem ~ 590–620 nm) in order to create variants that exhibit either long Stokes shift fluorescence
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