FRET protease assays for botulinum serotype A/E toxins
AssigneeAllergan, Inc.
InventorsLance E. Steward, Ester Fernandez-Salas, Kei Roger Aoki
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Abstract
The present invention provides clostridial toxin substrates useful in assaying for the protease activity of any clostridial toxin, including botulinum toxins of all serotypes as well as tetanus toxins. A clostridial toxin substrate of the invention contains a donor fluorophore; an acceptor having an absorbance spectrum overlapping the emission spectrum of the donor fluorophore; and a clostridial toxin recognition sequence that includes a cleavage site, where the cleavage site intervenes between the donor fluorophore and the acceptor and where, under the appropriate conditions, resonance energy transfer is exhibited between the donor fluorophore and the acceptor.
Claims41
A method of determining protease activity of botulinum toxin serotype E (BoNT/E), comprising the steps of:
- (a) treating a BoNT/E substrate with a sample under conditions suitable for clostridial toxin protease activity, said BoNT/E substrate comprising
- (i) a donor fluorophore;
- (ii) an acceptor having an absorbance spectrum overlapping the emission spectrum of said donor fluorophore;
- and
- (iii) a BoNT/E recognition sequence comprising a BoNT/E P5-P4-P3-P2-P1-P1′-P2′-P3′-P4′-P5′ cleavage site sequence, said BoNT/E P5-P4-P3-P2-P1-P1′-P2′-P3′-P4′-P5′ cleavage site sequence intervening between said donor fluorophore and said acceptor;
- wherein, under the appropriate conditions, resonance energy transfer is exhibited between said donor fluorophore and said acceptor;
- (b) exciting said donor fluorophore;
- and
- (c) determining resonance energy transfer of said treated substrate relative to a control substrate, wherein a difference in resonance energy transfer of said treated substrate as compared to said control substrate is indicative of BoNT/E protease activity.
A method of determining protease activity of botulinum toxin serotype E (BoNT/E), comprising the steps of:
- (a) treating a BoNT/E substrate with a sample under conditions suitable for clostridial toxin protease activity, said BoNT/E substrate comprising
- (i) a donor fluorophore;
- (ii) an acceptor having an absorbance spectrum overlapping the emission spectrum of said donor fluorophore;
- and
- (iii) a BoNT/E recognition sequence comprising a BoNT/E P5-P4-P3-P2-P1-P1′-P2′-P3′-P4′-P5′ cleavage site sequence including a BoNT/E P1-P1′ cleavage site, said BoNT/E P1-P1′ cleavage site intervening between said donor fluorophore and said acceptor;
- wherein either said donor fluorophore or said acceptor is not positioned within said BoNT/E P5-P4-P3-P2-P1-P1′-P2′-P3′-P4′-P5′ cleavage site sequence;
- and
- wherein, under the appropriate conditions, resonance energy transfer is exhibited between said donor fluorophore and said acceptor;
- (b) exciting said donor fluorophore;
- and
- (c) determining resonance energy transfer of said treated substrate relative to a control substrate, wherein a difference in resonance energy transfer of said treated substrate as compared to said control substrate is indicative of BoNT/E protease activity.
A method of determining protease activity of botulinum toxin serotype E (BoNT/E), comprising the steps of:
- (a) treating a BoNT/E substrate with a sample under conditions suitable for clostridial toxin protease activity, said BoNT/E substrate comprising
- (i) a donor fluorophore;
- (ii) an acceptor having an absorbance spectrum overlapping the emission spectrum of said donor fluorophore;
- and
- (iii) a BoNT/E recognition sequence comprising a BoNT/E P5-P4-P3-P2-P1-P1′-P2′-P3′-P4′-P5′ cleavage site sequence, said BoNT/E P5-P4-P3-P2-P1-P1′-P2′-P3′-P4′-P5′ cleavage site sequence intervening between said donor fluorophore and said acceptor;
- wherein either of said donor fluorophore, said acceptor, or both said donor fluorophore and said acceptor are genetically encoded;
- and
- wherein, under the appropriate conditions, resonance energy transfer is exhibited between said donor fluorophore;
- (b) exciting said donor fluorophore;
- and
- (c) determining resonance energy transfer of said treated substrate relative to a control substrate, wherein a difference in resonance energy transfer of said treated substrate as compared to said control substrate is indicative of BoNT/E protease activity.
Dependencies are inferred from the claim text.