title: Genetically encoded protease substrate based on lanthanide-binding peptide for time-gated fluorescence detection.
abstract: The study of biomolecular interactions is at the heart of biomedical research. Fluorescence and Förster resonance energy transfer (FRET) are potent and versatile tools in studying these interactions. Fluorescent proteins enable genetic encoding which facilitates their use in recombinant protein and in vivo applications. To eliminate the autofluorescence background encountered in applications based on fluorescent proteins, lanthanide labels can be used as donor fluorophores. Their long emission lifetime enables the use of time-gating that significantly improves assay sensitivity. In this work, we have combined the favorable characteristics of a terbium-ion-containing lanthanide-binding peptide (Tb(3+)-LBP) and green fluorescent protein (GFP) in a FRET-based homogeneous protease activity assay. The used genetically engineered construct had LBP and GFP sequences at adjacent ends of a linker that encoded the recognition sequence for caspase-3. Caspase proteases are central mediators in apoptosis and, consequently, are of great interest in the pharmaceutical industry. The designed fluorogenic protease substrate was applied for the detection of caspase-3 activity. We were able to demonstrate, for the first time, the applicability of a Tb(3+)-LBP-GFP energy-transfer pair in a protease activity assay. The intrinsically fluorescent and genetically encodable components enable easy expression of the construct without the need of cumbersome chemical labeling. By varying the fluorescent protein and the protease specificity of the internal linker sequence, the method can be applied for the detection of a wide variety of proteases.
authors.affiliation: Department of Biotechnology, University of Turku, Tykistökatu 6A, FI-20520 Turku, Finland. johanna.vuojola@utu.fi
authors.forename: Johanna
authors.initials: J
authors.lastname: Vuojola
authors.forename: Markku
authors.initials: M
authors.lastname: Syrjänpää
authors.forename: Urpo
authors.initials: U
authors.lastname: Lamminmäki
authors.forename: Tero
authors.initials: T
authors.lastname: Soukka
mesh_terms: D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D028581:Lanthanoid Series Elements / Q000032:analysis / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D028581:Lanthanoid Series Elements; D049452:Green Fluorescent Proteins; D010447:Peptide Hydrolases