Abstract: The present invention provides a method of determining the presence or activity of a clostridial toxin by (a) treating with a sample, under conditions suitable for clostridial toxin protease activity, a clostridial toxin substrate which includes a fluorophore; a bulking group; and a clostridial toxin recognition sequence containing a cleavage site that intervenes between the fluorophore and the bulking group; (b) exciting the fluorophore with plane polarized light; and (c) determining fluorescence polarization of the treated substrate relative to a control substrate, where a change in fluorescence polarization of the treated substrate as compared to fluorescence polarization of the control substrate is indicative of the presence or activity of the clostridial toxin.

Abstract: The present invention provides a method of producing an evolved clostridial toxin light chain having altered protease specificity by (a) generating a population, each member of which contains a clostridial toxin light chain variant or functional fragment thereof; (b) assaying the population for protease activity towards a selected clostridial toxin-resistant target protein, where increased protease activity is indicative of an evolved clostridial toxin light chain; and (c) isolating from the population one or more members, which contain an evolved clostridial toxin light chain or functional fragment thereof. Also provided herein are compositions which contain an evolved clostridial toxin light chain or functional fragment thereof having altered protease specificity.