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: 6.65). 11/2008; 15(10):1116-24. DOI: 10.1016/j.chembiol.2008.08.006
Source: PubMed


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,
  • Source
    • "BFP was further modified to develop a stable red fluorescent protein (RFP). Site directed and random mutagenesis of RFP generated mTagBFP with a shift in excitation spectrum and blue emission [36]. Although mTagBFP is the brightest among all the BFPs produced, it is 1.4-fold less photostable than EBFP2 [23]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Live cell imaging enables the study of dynamic processes of living cells in real time by use of suitable reporter proteins and the staining of specific cellular structures and/or organelles. With the availability of advanced optical devices and improved cell culture protocols it has become a rapidly growing research methodology. The success of this technique relies mainly on the selection of suitable reporter proteins, construction of recombinant plasmids possessing cell type specific promoters as well as reliable methods of gene transfer. This review aims to provide an overview of the recent developments in the field of marker proteins (bioluminescence and fluorescent) and methodologies (fluorescent resonance energy transfer, fluorescent recovery after photobleaching and proximity ligation assay) employed as to achieve an improved imaging of biological processes in hepatoma cells. Moreover, different expression systems of marker proteins and the modes of gene transfer are discussed with emphasis on the study of lipid droplet formation in hepatocytes as an example.
    BMC Cell Biology 07/2014; 15(1):26. DOI:10.1186/1471-2121-15-26 · 2.34 Impact Factor
  • Source
    • "Alternative versions of pFluo-2A-ZFN, called pFP-ZFN, were created. First, pFP-ZFNL and pFP-ZFNR constructs were generated that encode green-fluorescent protein (GFP) (28,29) and red fluorescent protein (RFP) (30), respectively, linked to ZFN via a 2A sequence (Supplementary Figure S7). Fluorescent proteins and ZFNs can be swapped using the NsiI and KpnI/XhoI restriction sites, respectively. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Targeted endonucleases including zinc finger nucleases (ZFNs) and clustered regularly interspaced short palindromic repeats (CRISPRs)/Cas9 are increasingly being used for genome editing in higher species. We therefore devised a broadly applicable and versatile method for increasing editing efficiencies by these tools. Briefly, 2A peptide-coupled co-expression of fluorescent protein and nuclease was combined with fluorescence-activated cell sorting (FACS) to allow for efficient isolation of cell populations with increasingly higher nuclease expression levels, which translated into increasingly higher genome editing rates. For ZFNs, this approach, combined with delivery of donors as single-stranded oligodeoxynucleotides and nucleases as messenger ribonucleic acid, enabled high knockin efficiencies in demanding applications, including biallelic codon conversion frequencies reaching 30-70% at high transfection efficiencies and ∼2% at low transfection efficiencies, simultaneous homozygous knockin mutation of two genes with ∼1.5% efficiency as well as generation of cell pools with almost complete codon conversion via three consecutive targeting and FACS events. Observed off-target effects were minimal, and when occurring, our data suggest that they may be counteracted by selecting intermediate nuclease levels where off-target mutagenesis is low, but on-target mutagenesis remains relatively high. The method was also applicable to the CRISPR/Cas9 system, including CRISPR/Cas9 mutant nickase pairs, which exhibit low off-target mutagenesis compared to wild-type Cas9.
    Nucleic Acids Research 04/2014; 42(10). DOI:10.1093/nar/gku251 · 9.11 Impact Factor
  • Source
    • "In a final series of experiments, we asked whether we could manipulate the output of Co-Driver, i.e. the timing and cell-type specificity of Cre recombination, by producing Dre and Roxed-Cre from distinct tissue-specific expression constructs. To address this question, we cloned Dre and Roxed-Cre into a hGFAP-promoter (17) cassette and the Thy1.2 expression cassette (18) to generate hGFAP-Dre, hGFAP-Roxed-Cre, Thy1.2-Dre and Thy1.2-Roxed-Cre constructs [Figure 5A, here, Dre was fused to the porcine teschovirus-1 (P)2 A sequence (37) and the blue fluorescent protein, mTagBFP (38)]. hGFAP-Cre transgenic mice express Cre in radial glia, the principal VZ precursor cells, between E13 and E17 and in the adult brain in astrocytes but not in neurons or oligodendrocytes (39). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Conditional mutagenesis using Cre recombinase expressed from tissue specific promoters facilitates analyses of gene function and cell lineage tracing. Here, we describe two novel dual-promoter-driven conditional mutagenesis systems designed for greater accuracy and optimal efficiency of recombination. Co-Driver employs a recombinase cascade of Dre and Dre-respondent Cre, which processes loxP-flanked alleles only when both recombinases are expressed in a predetermined temporal sequence. This unique property makes Co-Driver ideal for sequential lineage tracing studies aimed at unraveling the relationships between cellular precursors and mature cell types. Co-InCre was designed for highly efficient intersectional conditional transgenesis. It relies on highly active trans-splicing inteins and promoters with simultaneous transcriptional activity to reconstitute Cre recombinase from two inactive precursor fragments. By generating native Cre, Co-InCre attains recombination rates that exceed all other binary SSR systems evaluated in this study. Both Co-Driver and Co-InCre significantly extend the utility of existing Cre-responsive alleles.
    Nucleic Acids Research 01/2014; 42(6). DOI:10.1093/nar/gkt1361 · 9.11 Impact Factor
Show more