The Experts below are selected from a list of 2415 Experts worldwide ranked by ideXlab platform

Robert E. Campbell - One of the best experts on this subject based on the ideXlab platform.

  • Engineering of mCherry variants with long Stokes shift, red-shifted fluorescence, and low cytotoxicity
    PloS one, 2017
    Co-Authors: Yi Shen, Nathan C. Shaner, Yingche Chen, Robert E. Campbell
    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.

  • A Tandem Green-Red Heterodimeric Fluorescent Protein with High FRET Efficiency.
    Chembiochem : a European journal of chemical biology, 2016
    Co-Authors: Matthew D. Wiens, Yi Shen, M. Alaraby Salem, Nick Smisdom, Wei Zhang, Alex Brown, Robert E. Campbell
    Abstract:

    The tetrameric red fluorescent protein from Discosoma sp. coral (DsRed) has previously been engineered to produce dimeric and monomeric fluorescent variants with excitation and emission profiles that span the visible spectrum. The brightest of the effectively monomeric DsRed variants is tdTomato-a tandem fusion of a dimeric DsRed variant. Here we describe the engineering of brighter red (RRvT), green (GGvT), and green-red heterodimeric (GRvT) tdTomato variants. GRvT exhibited 99 % intramolecular FRET efficiency, resulting in long Stokes shift red fluorescence. These new variants could prove useful for multicolor live-cell imaging applications.

  • An Engineered Monomeric Zoanthus sp. Yellow Fluorescent Protein
    Chemistry & biology, 2013
    Co-Authors: Hiofan Hoi, Wei Zhang, Elizabeth S. Howe, Yidan Ding, Michelle A. Baird, Brittney R. Sell, John R. Allen, Michael W. Davidson, Robert E. Campbell
    Abstract:

    Summary Protein engineering has created a palette of monomeric fluorescent proteins (FPs), but there remains an ∼30 nm spectral gap between the most red-shifted useful Aequorea victoria green FP (GFP) variants and the most blue-shifted useful Discosoma sp . red FP (RFP) variants. To fill this gap, we have engineered a monomeric version of the yellow FP (YFP) from Zoanthus sp . coral. Our preferred variant, designated as mPapaya1, displays excellent fluorescent brightness, good photostability, and retains its monomeric character both in vitro and in living cells in the context of protein chimeras. We demonstrate that mPapaya1 can serve as a good Forster resonance energy transfer (FRET) acceptor when paired with an mTFP1 donor. mPapaya1 is a valuable addition to the palette of FP variants that are useful for multicolor imaging and FRET-based biosensing.

  • Dimerization-Dependent Green and Yellow Fluorescent Proteins
    ACS synthetic biology, 2012
    Co-Authors: Spencer C. Alford, Yidan Ding, Thomas Simmen, Robert E. Campbell
    Abstract:

    Dimerization-dependent fluorescent proteins (ddFP) are a recently introduced class of genetically encoded reporters that can be used for the detection of protein interactions in live cells. The progenitor of this class of tools was a red fluorescent ddFP (ddRFP) derived from a homodimeric variant of Discosoma red fluorescent protein. Here, we describe the engineering and application of an expanded palette of ddFPs, which includes green (ddGFP) and yellow (ddYFP) variants. These optimized variants offer several advantages relative to ddRFP including increased in vitro contrast and brightness for ddGFP and increased brightness and a lowered pKa for ddYFP. We demonstrate that both variants are useful as biosensors for protease activity in live cells. Using the ddGFP tool, we generated a highly effective indicator of endomembrane proximity that can be used to image the mitochondria-associated membrane (MAM) interface of endoplasmic reticulum (ER) and mitochondria.

  • Dimerization-Dependent Green and Yellow Fluorescent Proteins
    2012
    Co-Authors: Spencer C. Alford, Thomas Simmen, Yidan Ding, Robert E. Campbell
    Abstract:

    Dimerization-dependent fluorescent proteins (ddFP) are a recently introduced class of genetically encoded reporters that can be used for the detection of protein interactions in live cells. The progenitor of this class of tools was a red fluorescent ddFP (ddRFP) derived from a homodimeric variant of Discosoma red fluorescent protein. Here, we describe the engineering and application of an expanded palette of ddFPs, which includes green (ddGFP) and yellow (ddYFP) variants. These optimized variants offer several advantages relative to ddRFP including increased in vitro contrast and brightness for ddGFP and increased brightness and a lowered pKa for ddYFP. We demonstrate that both variants are useful as biosensors for protease activity in live cells. Using the ddGFP tool, we generated a highly effective indicator of endomembrane proximity that can be used to image the mitochondria-associated membrane (MAM) interface of endoplasmic reticulum (ER) and mitochondria

Nathan C. Shaner - One of the best experts on this subject based on the ideXlab platform.

  • Engineering of mCherry variants with long Stokes shift, red-shifted fluorescence, and low cytotoxicity
    PloS one, 2017
    Co-Authors: Yi Shen, Nathan C. Shaner, Yingche Chen, Robert E. Campbell
    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.

  • The mFruit Collection of Monomeric Fluorescent Proteins
    Clinical Chemistry, 2013
    Co-Authors: Nathan C. Shaner
    Abstract:

    Featured Article: Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, Tsien RY. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 2004:22;1567–72.2 Fluorescent proteins, starting with the green fluorescent protein (GFP)3 from Aequorea victoria, have revolutionized our ability to noninvasively study living systems (1). Practically every cell biologist has used fluorescent proteins to tag a favorite protein and watch its dynamic localization in living cells. For almost 10 years after the gene encoding GFP was cloned, researchers were limited to using only about half of the visible spectrum with these probes and were able to image only 2 different colors simultaneously, at best. In 1999, Mikhail Matz and colleagues at the Russian Academy of Science discovered the first known red fluorescent protein, in Discosoma sp. coral; that protein would become known as DsRed (2). Hopes were high that the wavelength barrier had been broken, and many researchers jumped at the chance to try this new color. Unfortunately, it soon …

  • improving the photostability of bright monomeric orange and red fluorescent proteins
    Nature Methods, 2008
    Co-Authors: Nathan C. Shaner, Paul Steinbach, Michael W. Davidson, Michael R. Mckeown, Kristin L. Hazelwood, Roger Y Tsien
    Abstract:

    Improved photostability of fluorescent proteins would benefit many applications but is usually an afterthought in selection screens. Setting photostability as the primary selection criterion in screens for improved fluorescent proteins yielded highly photostable variants of existing orange and red fluorescent proteins without compromising other beneficial characteristics. All organic fluorophores undergo irreversible photobleaching during prolonged illumination. Although fluorescent proteins typically bleach at a substantially slower rate than many small-molecule dyes, in many cases the lack of sufficient photostability remains an important limiting factor for experiments requiring large numbers of images of single cells. Screening methods focusing solely on brightness or wavelength are highly effective in optimizing both properties, but the absence of selective pressure for photostability in such screens leads to unpredictable photobleaching behavior in the resulting fluorescent proteins. Here we describe an assay for screening libraries of fluorescent proteins for enhanced photostability. With this assay, we developed highly photostable variants of mOrange (a wavelength-shifted monomeric derivative of DsRed from Discosoma sp.) and TagRFP (a monomeric derivative of eqFP578 from Entacmaea quadricolor) that maintain most of the beneficial qualities of the original proteins and perform as reliably as Aequorea victoria GFP derivatives in fusion constructs.

  • Improving the photostability of bright monomeric orange and red fluorescent proteins.
    Nature methods, 2008
    Co-Authors: Nathan C. Shaner, Paul Steinbach, Michael W. Davidson, Michael Z. Lin, Michael R. Mckeown, Kristin L. Hazelwood, Roger Y Tsien
    Abstract:

    All organic fluorophores undergo irreversible photobleaching during prolonged illumination. Although fluorescent proteins typically bleach at a substantially slower rate than many small-molecule dyes, in many cases the lack of sufficient photostability remains an important limiting factor for experiments requiring large numbers of images of single cells. Screening methods focusing solely on brightness or wavelength are highly effective in optimizing both properties, but the absence of selective pressure for photostability in such screens leads to unpredictable photobleaching behavior in the resulting fluorescent proteins. Here we describe an assay for screening libraries of fluorescent proteins for enhanced photostability. With this assay, we developed highly photostable variants of mOrange (a wavelength-shifted monomeric derivative of DsRed from Discosoma sp.) and TagRFP (a monomeric derivative of eqFP578 from Entacmaea quadricolor) that maintain most of the beneficial qualities of the original proteins and perform as reliably as Aequorea victoria GFP derivatives in fusion constructs.

  • improved monomeric red orange and yellow fluorescent proteins derived from Discosoma sp red fluorescent protein
    Nature Biotechnology, 2004
    Co-Authors: Nathan C. Shaner, Robert E. Campbell, Paul Steinbach, Ben N G Giepmans, Amy E Palmer, Roger Y Tsien
    Abstract:

    Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein

Roger Y Tsien - One of the best experts on this subject based on the ideXlab platform.

  • improving the photostability of bright monomeric orange and red fluorescent proteins
    Nature Methods, 2008
    Co-Authors: Nathan C. Shaner, Paul Steinbach, Michael W. Davidson, Michael R. Mckeown, Kristin L. Hazelwood, Roger Y Tsien
    Abstract:

    Improved photostability of fluorescent proteins would benefit many applications but is usually an afterthought in selection screens. Setting photostability as the primary selection criterion in screens for improved fluorescent proteins yielded highly photostable variants of existing orange and red fluorescent proteins without compromising other beneficial characteristics. All organic fluorophores undergo irreversible photobleaching during prolonged illumination. Although fluorescent proteins typically bleach at a substantially slower rate than many small-molecule dyes, in many cases the lack of sufficient photostability remains an important limiting factor for experiments requiring large numbers of images of single cells. Screening methods focusing solely on brightness or wavelength are highly effective in optimizing both properties, but the absence of selective pressure for photostability in such screens leads to unpredictable photobleaching behavior in the resulting fluorescent proteins. Here we describe an assay for screening libraries of fluorescent proteins for enhanced photostability. With this assay, we developed highly photostable variants of mOrange (a wavelength-shifted monomeric derivative of DsRed from Discosoma sp.) and TagRFP (a monomeric derivative of eqFP578 from Entacmaea quadricolor) that maintain most of the beneficial qualities of the original proteins and perform as reliably as Aequorea victoria GFP derivatives in fusion constructs.

  • Improving the photostability of bright monomeric orange and red fluorescent proteins.
    Nature methods, 2008
    Co-Authors: Nathan C. Shaner, Paul Steinbach, Michael W. Davidson, Michael Z. Lin, Michael R. Mckeown, Kristin L. Hazelwood, Roger Y Tsien
    Abstract:

    All organic fluorophores undergo irreversible photobleaching during prolonged illumination. Although fluorescent proteins typically bleach at a substantially slower rate than many small-molecule dyes, in many cases the lack of sufficient photostability remains an important limiting factor for experiments requiring large numbers of images of single cells. Screening methods focusing solely on brightness or wavelength are highly effective in optimizing both properties, but the absence of selective pressure for photostability in such screens leads to unpredictable photobleaching behavior in the resulting fluorescent proteins. Here we describe an assay for screening libraries of fluorescent proteins for enhanced photostability. With this assay, we developed highly photostable variants of mOrange (a wavelength-shifted monomeric derivative of DsRed from Discosoma sp.) and TagRFP (a monomeric derivative of eqFP578 from Entacmaea quadricolor) that maintain most of the beneficial qualities of the original proteins and perform as reliably as Aequorea victoria GFP derivatives in fusion constructs.

  • improved monomeric red orange and yellow fluorescent proteins derived from Discosoma sp red fluorescent protein
    Nature Biotechnology, 2004
    Co-Authors: Nathan C. Shaner, Robert E. Campbell, Paul Steinbach, Ben N G Giepmans, Amy E Palmer, Roger Y Tsien
    Abstract:

    Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein

  • improved monomeric red orange and yellow fluorescent proteins derived from Discosoma sp red fluorescent protein
    Nature Biotechnology, 2004
    Co-Authors: Nathan C. Shaner, Robert E. Campbell, Paul Steinbach, Ben N G Giepmans, Amy E Palmer, Roger Y Tsien
    Abstract:

    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.

Paul Steinbach - One of the best experts on this subject based on the ideXlab platform.

  • improving the photostability of bright monomeric orange and red fluorescent proteins
    Nature Methods, 2008
    Co-Authors: Nathan C. Shaner, Paul Steinbach, Michael W. Davidson, Michael R. Mckeown, Kristin L. Hazelwood, Roger Y Tsien
    Abstract:

    Improved photostability of fluorescent proteins would benefit many applications but is usually an afterthought in selection screens. Setting photostability as the primary selection criterion in screens for improved fluorescent proteins yielded highly photostable variants of existing orange and red fluorescent proteins without compromising other beneficial characteristics. All organic fluorophores undergo irreversible photobleaching during prolonged illumination. Although fluorescent proteins typically bleach at a substantially slower rate than many small-molecule dyes, in many cases the lack of sufficient photostability remains an important limiting factor for experiments requiring large numbers of images of single cells. Screening methods focusing solely on brightness or wavelength are highly effective in optimizing both properties, but the absence of selective pressure for photostability in such screens leads to unpredictable photobleaching behavior in the resulting fluorescent proteins. Here we describe an assay for screening libraries of fluorescent proteins for enhanced photostability. With this assay, we developed highly photostable variants of mOrange (a wavelength-shifted monomeric derivative of DsRed from Discosoma sp.) and TagRFP (a monomeric derivative of eqFP578 from Entacmaea quadricolor) that maintain most of the beneficial qualities of the original proteins and perform as reliably as Aequorea victoria GFP derivatives in fusion constructs.

  • Improving the photostability of bright monomeric orange and red fluorescent proteins.
    Nature methods, 2008
    Co-Authors: Nathan C. Shaner, Paul Steinbach, Michael W. Davidson, Michael Z. Lin, Michael R. Mckeown, Kristin L. Hazelwood, Roger Y Tsien
    Abstract:

    All organic fluorophores undergo irreversible photobleaching during prolonged illumination. Although fluorescent proteins typically bleach at a substantially slower rate than many small-molecule dyes, in many cases the lack of sufficient photostability remains an important limiting factor for experiments requiring large numbers of images of single cells. Screening methods focusing solely on brightness or wavelength are highly effective in optimizing both properties, but the absence of selective pressure for photostability in such screens leads to unpredictable photobleaching behavior in the resulting fluorescent proteins. Here we describe an assay for screening libraries of fluorescent proteins for enhanced photostability. With this assay, we developed highly photostable variants of mOrange (a wavelength-shifted monomeric derivative of DsRed from Discosoma sp.) and TagRFP (a monomeric derivative of eqFP578 from Entacmaea quadricolor) that maintain most of the beneficial qualities of the original proteins and perform as reliably as Aequorea victoria GFP derivatives in fusion constructs.

  • improved monomeric red orange and yellow fluorescent proteins derived from Discosoma sp red fluorescent protein
    Nature Biotechnology, 2004
    Co-Authors: Nathan C. Shaner, Robert E. Campbell, Paul Steinbach, Ben N G Giepmans, Amy E Palmer, Roger Y Tsien
    Abstract:

    Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein

  • improved monomeric red orange and yellow fluorescent proteins derived from Discosoma sp red fluorescent protein
    Nature Biotechnology, 2004
    Co-Authors: Nathan C. Shaner, Robert E. Campbell, Paul Steinbach, Ben N G Giepmans, Amy E Palmer, Roger Y Tsien
    Abstract:

    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.

Patrick C.h. Hsieh - One of the best experts on this subject based on the ideXlab platform.

  • Clearance kinetics of biomaterials affects stem cell retention and therapeutic efficacy.
    Biomacromolecules, 2014
    Co-Authors: Chia Y. Lai, Steve R. Roffler, Sho T. Lee, Shiaw M. Hwang, Shoei S. Wang, Kuan Wang, Patrick C.h. Hsieh
    Abstract:

    The use of biomaterial carriers to improve the therapeutic efficacy of stem cells is known to augment cell delivery, retention, and viability. However, the way that carrier clearance kinetics boosts stem cell delivery and impacts stem cell function remains poorly characterized. In this study, we designed a platform to simultaneously quantify carrier clearance and stem cell retention to evaluate the impact of carrier clearance kinetics on stem cell retention. Additionally, a murine model of hindlimb ischemia was employed to investigate the effects of various cell retention profiles on mitigating peripheral arterial disease. To image the in vivo behaviors of material and cells, we used biotinylated hyaluronan with fluorescently labeled streptavidin and Discosoma sp. Red (Ds-Red)-expressing human mesenchymal stem cells. We found that the retention of transplanted stem cells was closely related to the remaining biomaterial. Furthermore, therapeutic effectiveness was also affected by stem cell retention. These results demonstrate that low-molecular- weight hyaluronan had a slow clearance and high cell retention profile, improving the therapeutic efficacy of human stem cells.

  • Clearance Kinetics of Biomaterials Affects Stem Cell Retention and Therapeutic Efficacy
    2014
    Co-Authors: Chia Y. Lai, Steve R. Roffler, Sho T. Lee, Shoei S. Wang, Kuan Wang, Shiaw M. Hwang, Patrick C.h. Hsieh
    Abstract:

    The use of biomaterial carriers to improve the therapeutic efficacy of stem cells is known to augment cell delivery, retention, and viability. However, the way that carrier clearance kinetics boosts stem cell delivery and impacts stem cell function remains poorly characterized. In this study, we designed a platform to simultaneously quantify carrier clearance and stem cell retention to evaluate the impact of carrier clearance kinetics on stem cell retention. Additionally, a murine model of hindlimb ischemia was employed to investigate the effects of various cell retention profiles on mitigating peripheral arterial disease. To image the in vivo behaviors of material and cells, we used biotinylated hyaluronan with fluorescently labeled streptavidin and Discosoma sp. Red (Ds-Red)-expressing human mesenchymal stem cells. We found that the retention of transplanted stem cells was closely related to the remaining biomaterial. Furthermore, therapeutic effectiveness was also affected by stem cell retention. These results demonstrate that low-molecular-weight hyaluronan had a slow clearance and high cell retention profile, improving the therapeutic efficacy of human stem cells