Generation of Circularly Permuted Fluorescent-Protein-Based Indicators for In Vitro and In Vivo Detection of Citrate

Yuki Honda, Kotaro Kirimura

    Research output: Contribution to journalArticle

    4 Citations (Scopus)

    Abstract

    Indicators for citrate, particularly those applicable to its in vivo detection and quantitation, have attracted much interest in both biochemical studies and industrial applications since citrate is a key metabolic intermediate playing important roles in living cells. We generated novel fluorescence indicators for citrate by fusing the circularly permuted fluorescent protein (cpFP) and the periplasmic domain of the bacterial histidine kinase CitA, which can bind to citrate with high specificity. The ratiometric fluorescent signal change was observed with one of these cpFP-based indicators, named CF98: upon addition of citrate, the excitation peak at 504 nm increased proportionally to the decrease in the peak at 413 nm, suitable for build-in quantitative estimation of the binding compound. We confirmed that CF98 can be used for detecting citrate in vitro at millimolar levels in the range of 0.1 to 50 mM with high selectivity; even in the presence of other organic acids such as isocitrate and malate, the fluorescence intensity of CF98 remains unaffected. We finally demonstrated the in vivo applicability of CF98 to estimation of the intracellular citrate concentration in Escherichia coli co-expressing the genes encoding CF98 and the citrate carrier CitT. The novel indicator CF98 can be a specific and simple detection tool for citrate in vitro and a non-invasive tool for real-time estimation of intracellular concentrations of the compound in vivo.

    Original languageEnglish
    Article numbere64597
    JournalPLoS One
    Volume8
    Issue number5
    DOIs
    Publication statusPublished - 2013 May 22

    Fingerprint

    Citric Acid
    citrates
    Proteins
    Fluorescence
    Periplasmic Proteins
    fluorescence
    Gene encoding
    histidine kinase
    In Vitro Techniques
    fluorescent proteins
    Organic acids
    industrial applications
    Histidine
    malates
    Escherichia coli
    Industrial applications
    organic acids and salts
    Phosphotransferases
    Cells
    Acids

    ASJC Scopus subject areas

    • Agricultural and Biological Sciences(all)
    • Biochemistry, Genetics and Molecular Biology(all)
    • Medicine(all)

    Cite this

    Generation of Circularly Permuted Fluorescent-Protein-Based Indicators for In Vitro and In Vivo Detection of Citrate. / Honda, Yuki; Kirimura, Kotaro.

    In: PLoS One, Vol. 8, No. 5, e64597, 22.05.2013.

    Research output: Contribution to journalArticle

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