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

A method of identifying a first protease with substrate specificity for a non-neuronal SNARE protein, comprising:

• providing a first reporting construct comprising a first non-neuronal SNARE protein or a fragment thereof that comprises a protease cleavage site of the first non-neuronal SNARE protein, interposed between a first reporter and a second reporter,
• wherein the first non-neuronal SNARE protein or fragment thereof is dimensioned to provide an energy transfer between the first reporter and the second reporter to generate a first signal;
• providing a library comprising a plurality of proteases each comprising a mutated botulinum toxin light chain peptide, wherein each of the plurality of proteases has a different non-native exosite recognition sequence;
• contacting individual proteases of the library with the first reporting construct;
• and
• characterizing a first signal obtained from the first reporting construct,
• wherein change of the first signal indicates identification of a first protease having substrate specificity for the first non-neuronal SNARE protein.