Abstract
Genetically-encoded fluorescence resonance energy transfer (FRET) sensors for phosphate (P(i)) (FLIPPi) were engineered by fusing a predicted Synechococcus phosphate-binding protein (PiBP) to eCFP and Venus. Purified fluorescent indicator protein for inorganic phosphate (FLIPPi), in which the fluorophores are attached to the same PiBP lobe, shows P(i)-dependent increases in FRET efficiency. FLIPPi affinity mutants cover P(i) changes over eight orders of magnitude. COS-7 cells co-expressing a low-affinity FLIPPi and a Na(+)/P(i) co-transporter exhibited FRET changes when perfused with 100 microM P(i), demonstrating concentrative P(i) uptake by PiT2. FLIPPi sensors are suitable for real-time monitoring of P(i) metabolism in living cells, providing a new tool for fluxomics, analysis of pathophysiology or changes of P(i) during cell migration.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Animals
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Bacterial Proteins / chemistry
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Bacterial Proteins / genetics
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Bacterial Proteins / metabolism
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Base Sequence
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Binding Sites / genetics
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Biosensing Techniques / methods*
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CHO Cells
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COS Cells
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Chlorocebus aethiops
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Cricetinae
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DNA, Bacterial / genetics
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Fluorescence Resonance Energy Transfer / methods*
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Fluorescent Dyes
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Green Fluorescent Proteins / genetics
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Green Fluorescent Proteins / metabolism
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Kinetics
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Luminescent Proteins / genetics
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Luminescent Proteins / metabolism
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Models, Molecular
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Mutagenesis, Site-Directed
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Phosphate-Binding Proteins / chemistry
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Phosphate-Binding Proteins / genetics
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Phosphate-Binding Proteins / metabolism
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Phosphates / metabolism*
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Protein Conformation
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Protein Engineering
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Sequence Deletion
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Synechococcus / genetics
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Synechococcus / metabolism
Substances
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Bacterial Proteins
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Cyan Fluorescent Protein
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DNA, Bacterial
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Fluorescent Dyes
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Luminescent Proteins
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Phosphate-Binding Proteins
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Phosphates
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Recombinant Fusion Proteins
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yellow fluorescent protein, Bacteria
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Green Fluorescent Proteins