Document Detail


Genetically Encoded Protease Substrate Based on Lanthanide-Binding Peptide for Time-Gated Fluorescence Detection.
MedLine Citation:
PMID:  23272697     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
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 (Tb3+-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 are consequently of great interest in 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 Tb3+-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:
Johanna Vuojola; Markku Syrjänpää; Urpo Lamminmäki; Tero Soukka
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-12-31
Journal Detail:
Title:  Analytical chemistry     Volume:  -     ISSN:  1520-6882     ISO Abbreviation:  Anal. Chem.     Publication Date:  2012 Dec 
Date Detail:
Created Date:  2012-12-31     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0370536     Medline TA:  Anal Chem     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
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