US 12,287,323 B2
Reporting construct with synaptobrevin based moiety
Timothy Piazza, McFarland, WI (US); Francis Mark Dunning, Madison, WI (US); and Ward C Tucker, Monona, WI (US)
Assigned to BIOMADISON, INC., Del Mar, CA (US)
Filed by BIOMADISON, INC., Del Mar, CA (US)
Filed on Jan. 25, 2022, as Appl. No. 17/583,594.
Application 17/583,594 is a division of application No. 15/597,002, filed on May 16, 2017, granted, now 12,019,066.
Claims priority of provisional application 62/404,513, filed on Oct. 5, 2016.
Claims priority of provisional application 62/336,964, filed on May 16, 2016.
Prior Publication US 2022/0196634 A1, Jun. 23, 2022
Int. Cl. G01N 33/50 (2006.01); C07K 14/435 (2006.01); C07K 14/705 (2006.01); C12Q 1/37 (2006.01); G06T 7/00 (2017.01)
CPC G01N 33/5005 (2013.01) [C07K 14/43595 (2013.01); C07K 14/705 (2013.01); C12Q 1/37 (2013.01); G06T 7/001 (2013.01); G06T 7/0016 (2013.01); C07K 2319/50 (2013.01); C07K 2319/60 (2013.01); C07K 2319/70 (2013.01); G01N 2333/33 (2013.01); G01N 2333/952 (2013.01); G06T 2207/30072 (2013.01); G06T 2207/30242 (2013.01)] 11 Claims
 
1. A pair of reporting constructs for characterizing botulinum serotype neurotoxin comprising:
a first reporter construct comprising a first membrane binding peptide configured to localize to a vesicle membrane and a first fluorescent peptide joined by a first linking peptide interposed between the first membrane binding peptide and the first fluorescent peptide, wherein the first fluorescent peptide has a first emission wavelength, and wherein the first linking peptide is derived from synaptobrevin; and
a second reporter construct comprising a second membrane binding peptide configured to localize to the vesicle membrane and a second fluorescent peptide joined by a second linking peptide interposed between the second membrane binding peptide and the second fluorescent peptide, wherein the second fluorescent peptide has a second emission wavelength, and wherein the second linking peptide is derived from synaptobrevin,
wherein the first fluorescent peptide and the second fluorescent peptide are positioned such that less than 5% Forster resonance energy transfer (FRET) occurs between them when the first reporting construct and the second reporting construct are co-located on a membrane surface of the vesicle, and
wherein the first fluorescent peptide comprises one or more first mutations that increase the rate of intracellular proteolysis relative to an analogous fluorescent peptide that does not incorporate the one or more first mutations.