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

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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    • "Nonetheless, FPs have shown great promise for in vivo optoacoustic imaging in zebrafish [9] [20], glioma visualization [8], multi-contrast optoacoustic imaging [21] and optoacoustic flow cytometry [22]. Many fluorescent proteins lack photostability [16] [18], which may impede quantitative measurements [23]. To this end, nanosecond-laser-induced photobleaching has been shown to occur in isolated FPs [24]. "
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    ABSTRACT: Nanosecond-duration laser pulses are exploited in a plethora of therapeutic and diagnostic applications, such as optoacoustic imaging. However, phototoxicity effects of pulsed radiation in living cells, in particular those expressing genetic reporters, are not well understood. We established a three-dimensional fluorescent protein expressing cellular model in order to reliably investigate the extent and major exposure parameters responsible for both photobleaching and phototoxicity under pulsed laser exposure, unveiling a variety of possible effects on living cells, from reversible photobleaching to cytotoxicity and cell death. Significant losses of fluorescence levels were identified when exposing the cells to illumination conditions considered safe under common standards for skin exposure in diagnostic imaging applications. Thus, the use of photolabile fluorescent proteins and their in vivo exposure parameters have to be designed carefully for all applications using pulsed nanosecond radiation. In particular, loss of signal due to bleaching may significantly alter signals in longitudinal measurements, making data quantification challenging. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Biomaterials 08/2015; 69:38-44. DOI:10.1016/j.biomaterials.2015.07.051 · 8.56 Impact Factor
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    • "Red fluorescent protein mCherry is synthetically derived from successive modifications of the Discosoma sp. fluorescent protein DsRed (Shaner et al., 2004). Teal fluorescent protein mTFP1 is a synthetic derivative from the Cyan protein cF_48 from Clavularia soft coral (Ai et al., 2006). "
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    ABSTRACT: Despite the powerful potential of fluorescent proteins for labeling bacteria, their use has been limited in multispecies oral biofilm models. Fermentative metabolism by streptococcal species that initiate biofilm colonization results in an acidic, reduced microenvironment that may limit the activities of some fluorescent proteins which are influenced by pH and oxygen availability. The need to reliably distinguish morphologically similar strains within biofilms was the impetus for this work. Teal fluorescent protein (mTFP1) and red fluorescent protein (mCherry) were chosen because their fluorescent properties made them promising candidates. Since tRNA availability has been implicated in efficient translation of sufficient quantities of protein for maximum fluorescence, a streptococcal codon optimization approach was used. DNA was synthesized to encode either protein using codons most frequently used in streptococci; each coding region was preceded by an engineered ribosomal binding site and restriction sites for cloning a promoter. Plasmids carrying this synthesized DNA under control of the Streptococcus mutans lactate dehydrogenase promoter conferred fluorescence to nine representative streptococcal and two Enterococcus faecalis strains. Further characterization in Streptococcus gordonii showed that mTFP1 and mCherry expression could be detected in cells grown planktonically, in biofilms, or in colonies on agar when expressed on an extrachromosomal plasmid or in single copy integrated into the chromosome. This latter property facilitated counterselection of chromosomal mutations demonstrating value for bacterial strain construction. Fluorescent and non-fluorescent bacteria were distinguishable at acidic pH. These codon-optimized versions of mTFP1 and mCherry have promising potential for use in multiple experimental applications. Copyright © 2015. Published by Elsevier B.V.
    Journal of microbiological methods 06/2015; 116. DOI:10.1016/j.mimet.2015.06.010 · 2.03 Impact Factor
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    • "Our method relies on the expression of recombinant constructs labeled with fluorescent proteins, which are used as epitopes for the binding of small high-affinity binders, so-called nanobodies , as probes. By making use of the fluorescent proteins GFP or YFP and mHoneydew [28] which are of different evolutionary descent but possess similar spectral properties , we free the orange-red and far red parts of the spectrum to be used for bright organic dyes coupled to the nanobodies, making our assay easily accessible in Dual color single particle tracking via nanobodies "
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    ABSTRACT: Single particle tracking is a powerful tool to investigate the function of biological molecules by following their motion in space. However, the simultaneous tracking of two different species of molecules is still difficult to realize without compromising the length or density of trajectories, the localization accuracy or the simplicity of the assay. Here, we demonstrate a simple dual color single particle tracking assay using small, bright, high-affinity labeling via nanobodies of accessible targets with widely available instrumentation. We furthermore apply a ratiometric step-size analysis method to visualize differences in apparent membrane viscosity.
    06/2015; 3(2):1-8. DOI:10.1088/2050-6120/3/2/024001
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