Biomadison, Inc.

Francis Mark Dunning

Ward C Tucker

Timothy M Piazza

Recombinant nucleic acids encoding reporting constructs for characterizingneurotoxin protease activity and cells that incorporate such recombinant nucleic acids and that are suitable for use in cell-based assays for the neurotoxin are provided. The encoded reporting constructs are a pair of recombinant hybrid proteins that act in concert, and can include an N-terminal palmitoylation sequence. The reporting constructs are a pair of recombinant hybrid proteins that act in concert, and that include aneurotoxin protease recognition and cleavage sequence positioned to release a fluorophore upon cleavage.

Peptides for characterization ofneurotoxins

Compositions and methods for improved cell-based methods of characterizing botulinum neurotoxins are provided. Cells utilized in these methods include a reporting construct that is cleaved following uptake and processing of botulinum neurotoxin by the cell, resulting in proteolysis of the portion of the reporting construct that is released following cleavage. The released portion includes a fluorophore and amino acid substitutions or sequences that enhance the rate of proteolysis. A pair of reporting constructs can be utilized in which one member of the pair is modified to resist cleavage by the botulinum neurotoxin while co-localizing with the remaining member of the pair.

Reporting construct with synaptobrevin based moiety

Systems are described in which a primary detergent or surfactant in an aqueous solution is removed by the addition of a secondary detergent or surfactant in concentrations that exceed the critical micellar concentration (CMC) of the secondary detergent or surfactant using a size separation device. These systems are particularly applicable to protein-containing solutions. Typical primary detergents/surfactants include polysorbate 20, polysorbate 80, and Triton X-100. Suitable secondary detergents or surfactants can be ionic, nonionic, or zwitterionic. Typical secondary detergents/surfactants include, but are not limited to, galactoside detergents (e.g. octyl-β-galactoside), glucamide detergents (e.g. MEGA 8, MEGA 9, MEGA 10), cholamide detergents (e.g. CHAPS, CHAPSO, BIGCHAPS), and sulfobetaine detergents (such as sulfobetaine 3-10).

Systems for removal of detergents from aqueous solutions

Recombinant nucleic acids encoding reporting constructs for characterizingneurotoxin protease activity and cells that incorporate such recombinant nucleic acids and that are suitable for use in cell-based assays for the neurotoxin are provided. The encoded reporting constructs are a pair of recombinant hybrid proteins that act in concert. The reporting constructs are a pair of recombinant hybrid proteins that act in concert, and that include aneurotoxin protease recognition and cleavage sequence positioned to release a fluorophore upon cleavage.

Cells for characterization of botulinum neurotoxins

Assay with synaptobrevin based moiety

Vesicles that incorporate reporting constructs for characterizing Botulinum neurotoxin protease activity and suitable for use in an assay are provided. The reporting constructs are a pair of recombinant hybrid proteins that act in concert. The reporting constructs are a pair of recombinant hybrid proteins that act in concert, and that include a Botulinum neurotoxin protease recognition and cleavage sequence positioned to release a fluorophore upon cleavage.

Vesicles for characterization of botulinum neurotoxins

Compositions and methods are described in which a primary detergent or surfactant in an aqueous solution is removed by the addition of a secondary detergent or surfactant in concentrations that exceed the critical micellar concentration (CMC) of the secondary detergent or surfactant. These compositions and methods are particularly applicable to protein-containing solutions. Typical primary detergents/surfactants include polysorbate 20, polysorbate 80, and Triton X-100. Suitable secondary detergents or surfactants can be ionic, nonionic, or zwitterionic. Typical secondary detergents/surfactants include, but are not limited to, galactoside detergents (e.g. octyl-&#x3b2;-galactoside), glucamide detergents (e.g. MEGA 8, MEGA 9, MEGA 10), cholamide detergents (e.g. CHAPS, CHAPSO, BIGCHAPS), and sulfobetaine detergents (such as sulfobetaine 3-10).

Compositions and methods for removal of detergents from aqueous solutions

A protease directed to a non-neuronal SNARE protein is described. The protease is produced by selective mutation of aneurotoxin light chain, and is characterized utilizing a reporting construct that includes all or part of the non-neuronal SNARE protein. Such a protease has utility in the treatment of diseases associated with hypersecretion, where the hypersecretion is mediated by a non-neuronal SNARE protein.

Botulinum neurotoxins with modified light chain specificity and methods for producing same

Reporting constructs for characterizingneurotoxin protease activity and suitable for use in a cell-based assay are provided. The reporting construct can be a single recombinant hybrid protein or a pair of recombinant hybrid proteins that act in concert. The recombinant hybrid protein(s) include a fluorophore and an N-terminal non-peptide membrane anchoring portion. The recombinant hybrid protein or at least one of a pair of recombinant hybrid proteins that act in concert include aneurotoxin protease recognition and cleavage sequence positioned to release a fluorophore upon cleavage.

Reporting constructs for characterization ofneurotoxins

Compositions and methods for analyzing protease activity, and especially BoNT/B, BoNT/G, BoNT/D, and/or BoNT/F protease activity, using a cell based assay 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. The fluorophores are positioned so that no useful FRET occurs between them, permitting fluorescence produced by the non-released fluorophore to be used in data normalization.

Non-FRET cell based assay

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.

Growing community of inventors

Madison, WI, United States of America

Francis Mark Dunning