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US 10,246,492 B2Granted
Botulinum assay with synaptobrevin substrate moiety
AssigneeBioMadison, Inc.
InventorsWard C. Tucker, Francis Mark Dunning
Life of this patent
Estimating the term…
Abstract
Compositions and methods for analyzing intracellular BoNT protease activity, and especially BoNT/B, BoNT/G, BoNT/D, and/or BoNT/F protease activity are provided. Cells express at least two recombinant hybrid proteins each of which includes a fluorophore and a membrane anchoring peptide, and at least one of which includes a BoNT protease recognition and cleavage sequence positioned to release a fluorophore upon cleavage. Analysis is performed by monitoring fluorescence following exposure to a BoNT.
Claims11
A recombinant nucleic acid comprising:
- a sequence that encodes a first hybrid proteins having a structure of A-C-B and a second hybrid protein having a structure of A-C′ D;
- wherein A is a transmembrane protein domain of synaptobrevin targeted to an intracellular vesicle membrane and is not cleavable by a Botulinum neurotoxin protease, B is a first fluorescent protein, C is a first linking domain comprising a Botulinum neurotoxin protease recognition sequence and a Botulinum neurotoxin protease cleavage sequence, C′ is a second linking domain comprising an analog of C that includes the Botulinum neurotoxin protease recognition sequence but not the Botulinum neurotoxin protease cleavage sequence, and D is a second fluorescent protein selected to form a Förster resonance energy transfer (FRET) pair with the first fluorescent protein, wherein said transmembrane domain, said peptide spacer, and said BoNT protease recognition and cleavage sequence are selected to support FRET between said first fluorescent protein and said second fluorescent protein when said first hybrid protein and said second hybrid protein are collocated with a vesicle.
A transfected cell comprising:
- a nucleic acid comprising a sequence that encodes a first hybrid protein having a structure of A-C-B and a second hybrid protein having a structure of A-C′-D;
- wherein A is a transmembrane protein domain of synaptobrevin targeted to an intracellular vesicle membrane, and that is not cleavable by a Botulinum neurotoxin protease, B is a first fluorescent protein, C is a first linking region comprising a Botulinum neurotoxin protease recognition sequence and Botulinum neurotoxin protease cleavage sequence, C′ is a second linking region comprising an analog of C that includes the Botulinum neurotoxin protease recognition sequence but not the Botulinum neurotoxin protease cleavage sequence, and D is a second fluorescent protein selected to form a FRET pair with the first fluorescent protein, wherein said transmembrane domain, said peptide space, and said BoNT protease recognition and cleavage sequence are selected to support FRET between said first fluorescent protein and said second fluorescent protein when said first hybrid protein and said second hybrid protein are collated with a vesicle.
Dependencies are inferred from the claim text.