Methods of activating clostridial toxins

Abstract

The specification discloses modified Clostridial toxins comprising an exogenous Clostridial toxin di-chain loop protease cleavage site located within the di-chain loop region; polynucleotide molecules encoding such modified Clostridial toxins; method of producing such modified Clostridial toxins, method of activating such modified Clostridial toxins and methods of activating recombinantly-expressed Clostridial toxins.

What is claimed: 
     
         1 . A method of activating a modified Clostridial toxin, the method comprising the step of incubating a modified Clostridial toxin with a BoNT/A di-chain loop protease under physiological conditions;
 wherein the BoNT/A toxin di-chain loop protease is selected from the group consisting of SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO: 38;   wherein the modified Clostridial toxin comprises an exogenous BoNT/A di-chain loop protease cleavage site and is selected from the group consisting of a modified BoNT/B, a modified BoNT/C, a modified BoNT/D, a modified BoNT/E, a modified BoNT/F, a modified BoNT/G, a modified TeNT, a modified BaNT, a modified BuNT, and a Clostridial toxin chimeric variant; and   wherein cleavage of the modified Clostridial toxin by the BoNT/A di-chain loop protease converts the modified Clostridial toxin from its single-chain polypeptide form into its di-chain form, thereby activating the modified Clostridial toxin.   
     
     
         2 . The method of  claim 1 , wherein the modified Clostridial toxin is a modified BoNT/B. 
     
     
         3 . The method of  claim 1 , wherein the modified Clostridial toxin is a modified BoNT/C. 
     
     
         4 . The method of  claim 1 , wherein the modified Clostridial toxin is a modified BoNT/E. 
     
     
         5 . The method of  claim 1 , wherein the modified Clostridial toxin chimeric variant. 
     
     
         6 . A method of activating a recombinantly expressed Clostridial toxin, the method comprising the steps of:
 (a) expressing in a cell a polynucleotide molecule encoding a modified Clostridial toxin, wherein the modified Clostridial toxin comprises an exogenous BoNT/A di-chain loop protease cleavage site and is selected from the group consisting of a modified BoNT/B, a modified BoNT/C, a modified BoNT/D, a modified BoNT/E, a modified BoNT/F, a modified BoNT/G, a modified TeNT, a modified BaNT, a modified BuNT, and a Clostridial toxin chimeric variant;   (b) purifying the modified Clostridial toxin;   (c) incubating the purified modified Clostridial toxin with a BoNT/A di-chain loop protease under physiological conditions;   wherein the BoNT/A toxin di-chain loop protease is selected from the group consisting of SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO: 38; and   wherein cleavage of the modified Clostridial toxin by the BoNT/A di-chain loop protease converts the modified Clostridial toxin from its single-chain polypeptide form into its di-chain form, thereby activating the modified Clostridial toxin.