Shaner, N.C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567-1572

Department of Chemistry and Biochemistry, University of California, San Diego, San Diego, California, United States
Nature Biotechnology (Impact Factor: 41.51). 01/2005; 22(12):1567-72. DOI: 10.1038/nbt1037
Source: PubMed


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.

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Available from: Nathan C Shaner, Jan 21, 2015
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    • "Generation of GalR1-mCherry-[neo + ] and GalR2-hrGFP-[neo + ] knockin mice Mouse genomic clones including either GalR1 or GalR2 genes from the bMQ mouse strain 129S7 (129Sv) bacterial artificial chromosome (BAC) library (inserts 89–178 kb; Source BioScience; Adams et al., 2005) were electroporated into strain EL250 Escherichia coli (Lee et al., 2001). This allowed temperature-inducible, lambda Red-mediated, homologous recombination into the BAC (Copeland et al., 2001; Lee et al., 2001) of PCR products from either vector hrGFP-FRTneoFRT (Balthasar et al., 2004; Parton et al., 2007; hrGFP derived from Stratagene-Agilent vector phrGFP-1, Zeng et al., 2003; Fig. 2A, middle panel) flanked by GalR2 homologous sequence or vector mCherry- FRTneoFRT (hrGFP exchanged for mCherry; Shaner et al., 2004) flanked by GalR1 homologous sequence. Within the latter PCR product an AseI restriction site was introduced immediately downstream of the 3′ FRT site (Fig. 1A, middle panel), for use in Southern blot digests, and the mCherry/hrGFP heterologous 3′-untranslated region (UTR) has identity to nt 705–1193 of vector pCMV-Script (AF028239) which includes the SV40 early region poly(A) site (Connelly and Manley, 1988; J02400, nt 2828–2547). "
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    • "The mitochondrial localization of RRL–GFP was further confirmed by co-localization with a mitochondrion-selective dye MitoTracker Red (Fig. 1A–C). Additionally, the mitochondrial localization of RRL–GFP was also determined by tobacco leaf infiltration experiments with a well-known mitochondria marker, mt-rk CD3-991 (Nelson et al., 2007), which was generated with a red fluorescent protein, mCherry (Shaner et al., 2004). The RRL–GFP fusion protein co-localized with mt-rk CD3-991 by fluorescence analyses (Fig. 1D–F). "
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