US 2008/0166739 A1Published application

GFP-SNAP25 Fluorescence Release Assay for Botulinum Toxin Protease Activity

AssigneeAllergan, Inc.

InventorsLance E. Steward, Marcella A. Gilmore, Kei R. Aoki

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Abstract

The present invention provides a nucleic acid molecule which contains a nucleotide sequence encoding a SNAP-25 substrate which includes (i) a green fluorescent protein; (ii) a first partner of an affinity couple; and (iii) a portion of SNAP-25 that includes a BoNT/A, BoNT/C1 or BoNT/E recognition sequence containing a cleavage site, where the cleavage site intervenes between the green fluorescent protein and the first partner of the affinity couple. Further provided herein is a nucleic acid molecule which contains a nucleotide sequence encoding a tagged toxin substrate which includes (i) a fluorescent protein; (ii) a first partner of an affinity couple; and (iii) a clostridial toxin recognition sequence containing a cleavage site, where the cleavage site intervenes between the fluorescent protein and the first partner of the affinity couple.

Claims24

Dependent claims

A method of determining Botulinum toxin serotype A protease activity, comprising the steps of:

(a) treating with a sample, in solution phase under conditions suitable for Botulinum toxin serotype A protease activity, a tagged toxin substrate comprising
(i) a fluorescent protein;
(ii) a first partner of an affinity couple;
and
(iii) a Botulinum toxin serotype A recognition sequence comprising a cleavage site, where the cleavage site intervenes between said fluorescent protein and said first partner of the affinity couple, such that a fluorescent cleavage product is generated when Botulinum toxin serotype A is present in said sample;
(b) contacting said treated sample with a second partner of the affinity couple, thereby forming stable complexes comprising said first and second partners of said affinity couple;
and
(c) assaying the presence or amount of said fluorescent cleavage product in said treated sample, thereby determining Botulinum toxin serotype A protease activity.

5 more elements

Dependencies are inferred from the claim text.

Facts

Application: 12/018,637Filed 23 Jan 2008
Priority: 28 Aug 2001
Published: 10 Jul 2008
Term ends: Not estimatedThe source records don’t support an estimate.

Classification15 codes

C07K 14/705
From animals; from humans: receptors; Cell surface antigens; Cell surface determinants
C07K 14/001
Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof: by chemical synthesis
C07K 14/435
Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof: from animals; from humans
C07K 14/43595
From invertebrates: from coelenteratae, e.g. medusae

Family

Application09/942,098Continuation · filed 28 Aug 2001
Application10/917,844Division · filed 13 Aug 2004
Application12/018,637Division
Application10/917,844Continuation
US 2003/0143651 A1Published application
US 2005/0100973 A1Published application
US 2006/0154314 A9Published application
US 7,332,567 B2Related patent
US 7,374,896 B2Related patent
US 2008/0166739 A1This patent

From the US filing history. Foreign family members aren’t listed.