Conversion of Red Fluorescent Protein into a Bright Blue Probe

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Chemistry & Biology (Impact Factor: 6.59). 11/2008; 15(10):1116-24. DOI: 10.1016/j.chembiol.2008.08.006
Source: PubMed

ABSTRACT We used a red chromophore formation pathway, in which the anionic red chromophore is formed from the neutral blue intermediate, to suggest a rational design strategy to develop blue fluorescent proteins with a tyrosine-based chromophore. The strategy was applied to red fluorescent proteins of the different genetic backgrounds, such as TagRFP, mCherry, HcRed1, M355NA, and mKeima, which all were converted into blue probes. Further improvement of the blue variant of TagRFP by random mutagenesis resulted in an enhanced monomeric protein, mTagBFP, characterized by the substantially higher brightness, the faster chromophore maturation, and the higher pH stability than blue fluorescent proteins with a histidine in the chromophore. The detailed biochemical and photochemical analysis indicates that mTagBFP is the true monomeric protein tag for multicolor and lifetime imaging, as well as the outstanding donor for green fluorescent proteins in Förster resonance energy transfer applications.

Download full-text


Available from: Jinghang Zhang, Aug 09, 2015
  • Source
    • "Raeppli is a transgenic construct that contains the sequences necessary for marking cells with one of four possible fluorescent proteins that can be combinatorially used to provide multiple color combinations: TagBFP (Subach et al., 2008), mTFP1 (Ai et al., 2006), mOrange (Shaner et al., 2004) [or E2-Orange (Strack et al., 2009) for nuclear constructs] and mKate2 (Shcherbo et al., 2009) (Fig. 1A). These fluorescent proteins were chosen for their Fig. 1. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Observation of how cells divide, grow, migrate and form different parts of a developing organism is crucial for understanding developmental programs. Here, we describe a multicolor imaging tool named Raeppli (after the colorful confetti used at the carnival in Basel). Raeppli allows whole-tissue labeling such that the descendants of the majority of cells in a single organ are labeled and can be followed simultaneously relative to one another. We tested the use of Raeppli in the Drosophila melanogaster wing imaginal disc. Induction of Raeppli during larval stages irreversibly labels >90% of the cells with one of four spectrally separable, bright fluorescent proteins with low bias of selection. To understand the global growth characteristics of imaginal discs better, we induced Raeppli at various stages of development, imaged multiple fixed discs at the end of their larval development and estimated the size of their pouch primordium at those developmental stages. We also imaged the same wing disc through the larval cuticle at different stages of its development; the clones marked by Raeppli provide landmarks that can be correlated between multiple time points. Finally, we used Raeppli for continuous live imaging of prepupal eversion of the wing disc.
    Development 12/2013; 141(2). DOI:10.1242/dev.102913 · 6.27 Impact Factor
  • Source
    • "Following positive mCherry expression and detection , we tested the expression of five additional FPs that span the visual spectrum ( Table 1 ) . mTagBFP is a promising blue FP derived from the sea anemone Entacmaea quadri - color ( Subach et al . , 2008 ) . It is superior to the best Aequo - rea victoria blue fluorescent protein , EBFP2 , in terms of brightness , photostability , maturation kinetics and pH sen - sitivity , making it one of the most promising BFP color variants 12 for live - cell imaging available to date ( Rizzo et al . , 2009 ; Sample et al . , 2009 ) . mTagBFP perfor"
    [Show abstract] [Hide abstract]
    ABSTRACT: Fluorescent proteins (FPs) have become essential tools for a growing number of fields in biology. However, such tools have not been widely adopted for use in microalgal research. The aim of this study was to express and compare six different FPs, blue mTagBFP, cyan mCerulean, green CrGFP, yellow Venus, orange tdTomato, and red mCherry, in the popular model microalga Chlamydomonas reinhardtii. To circumvent transgene silencing that often occurs in C. reinhardtii, the FPs were expressed from the nuclear genome as transcriptional fusions with the sh-ble antibiotic resistance gene, with the foot-and-mouth-disease virus 2A self-cleaving sequence placed between the coding sequences. We demonstrate that all ble-2A-FPs tested are well-expressed and efficiently processed to yield mature, unfused FPs that localize throughout the cytoplasm. The fluorescence signals of each of the FPs were detectable in whole cells by fluorescence microplate reader analysis, live cell fluorescence microscopy, and flow cytometry. Furthermore, we report a comparative analysis of fluorescence levels over auto-fluorescence for the chosen FPs. Finally, we demonstrate that the ble-2A expression vector can be used to fluorescently label an endogenous protein, α-tubulin. We show that the mCerulean- α-tubulin fusion protein localizes to the cytoskeleton and flagella, as expected, and that cells containing this fusion protein had normal cellular function. Overall, our results indicate that, with the use of the ble-2A nuclear expression construct, a wide array of FP tools and technologies can now be applied to microalgal research, opening up many possibilities for microalgal biology and biotechnology. © 2013 The Authors. The Plant Journal © 2013 Blackwell Publishing Ltd.
    The Plant Journal 03/2013; 74(4). DOI:10.1111/tpj.12165 · 6.82 Impact Factor
  • Source
    • "This appeared to be due to extraordinarily high recombination rates leading to reporter loss and/or mis-expression (K.Dower, unpublished). As a result, a combination of more dissimilar fluorescent proteins such as Venus, Cherry, and TagBFP (Subach et al., 2008), derived from the sea anemone Entacmaea quadricolor, was required for stable, simultaneous incorporation. A useful aspect of the multi-reporter viruses was that early and intermediate reporter proteins showed saturating expression kinetics under standard conditions due to long reporter protein half-life. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Vaccinia virus is the prototypical orthopoxvirus of Poxviridae, a family of viruses that includes the human pathogens Variola (smallpox) and Monkeypox. Core viral functions are conserved among orthopoxviruses, and consequently Vaccinia is routinely used to study poxvirus biology and screen for novel antiviral compounds. Here we describe the development of a series of fluorescent protein-based reporter Vaccinia viruses that provide unprecedented resolution for tracking viral function. The reporter viruses are divided into two sets: (1) single reporter viruses that utilize temporally regulated early, intermediate, or late viral promoters; and (2) multi-reporter viruses that utilize multiple temporally regulated promoters. Promoter and reporter combinations were chosen that yielded high signal-to-background for stage-specific viral outputs. We provide examples for how these viruses can be used in the rapid and accurate monitoring of Vaccinia function and drug action.
    Antiviral research 07/2011; 91(1):72-80. DOI:10.1016/j.antiviral.2011.04.014 · 3.94 Impact Factor
Show more