Methods for the Manufacture of Proteolytically Processed Polypeptides

Abstract

The present invention relates to a novel proteolytically active polypeptide and various uses of the polypeptide (and others) in screening and manufacturing methods.

1 - 18 . (canceled) 
     
     
         19 . A composition comprising a proteolytically-processed polypeptide, the composition obtainable by a method comprising contacting a first polypeptide comprising an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 1 with a second polypeptide that is recombinant or purified and is susceptible to proteolysis by the first polypeptide, resulting in the proteolytic processing of the second polypeptide into at least two cleavage products, thereby providing the proteolytically-processed polypeptide. 
     
     
         20 - 22 . (canceled) 
     
     
         23 . The composition of  claim 19 , comprising a mixture of processed and unprocessed second polypeptide, the mixture containing less than 5% unprocessed second polypeptide. 
     
     
         24 . The composition of  claim 19 , comprising a mixture of processed and unprocessed second polypeptide, the mixture containing less than 3% unprocessed second polypeptide. 
     
     
         25 . The composition of  claim 19 , comprising a mixture of processed and unprocessed second polypeptide, the mixture containing less than 1% unprocessed second polypeptide. 
     
     
         26 . The composition of claim  9 , wherein the first polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1. 
     
     
         27 . The composition of  claim 19 , wherein the first polypeptide comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 1. 
     
     
         28 . The composition of  claim 19 , wherein the first polypeptide comprises an amino acid sequence that differs from SEQ ID NO: 1 by up to 10 conservative amino acid substitutions. 
     
     
         29 . The composition of  claim 19 , wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 1. 
     
     
         30 . The composition of  claim 19 , wherein the second polypeptide comprises an amino acid sequence haring at least 90% sequence identity with any one of SEQ ID NOs: 3 to 25. 
     
     
         31 . The composition of  claim 19 , wherein the second polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 3 to 25. 
     
     
         32 . The composition of  claim 30 , wherein the first polypeptide proteolytically cleaves the second polypeptide at a position immediately C-terminal to a basic amino acid residue within the amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 3 to 25. 
     
     
         33 . The composition of  claim 19 , wherein the second polypeptide is a clostridial neurotoxin or a modified clostridial neurotoxin. 
     
     
         34 . The composition of  claim 33 , wherein the second polypeptide is a recombinant clostridial neurotoxin or a modified recombinant clostridial neurotoxin. 
     
     
         35 . The composition of  claim 33 , wherein the second polypeptide is BoNT/A or a modified BoNT/A. 
     
     
         36 . The method of  claim 33 , wherein the modified clostridial neurotoxin: lacks a functional binding domain (H CC ) and is therefore not able to bind a native clostridial neurotoxin receptor; comprises a modified binding domain (H CC ) that binds to a native clostridial neurotoxin receptor; or comprises a non-clostridial binding domain that binds the modified clostridial neurotoxin to a non-native clostridial neurotoxin receptor. 
     
     
         37 . The composition of  claim 33 , wherein the modified clostridial neurotoxin is a chimeric clostridial neurotoxin. 
     
     
         38 . The composition of  claim 19 , wherein the second polypeptide is a single-chain clostridial neurotoxin or a modified single-chain clostridial neurotoxin prepared by recombinant expression in  E. coli.    
     
     
         39 . The composition of  claim 19 , wherein the second polypeptide is a single-chain clostridial neurotoxin and contact between the first and second polypeptides results in a clostridial neurotoxin di-chain polypeptide comprising a clostridial neurotoxin light chain that is covalently linked by a disulfide bond to a clostridial neurotoxin heavy chain. 
     
     
         40 . The composition of  claim 39 , wherein the C-terminus of the clostridial neurotoxin light chain and the N-terminus of the clostridial neurotoxin heavy chain are identical to the corresponding termini of a corresponding di-chain clostridial neurotoxin generated from the same single-chain clostridial neurotoxin polypeptide in wild-type  Clostridia.    
     
     
         41 . The composition of  claim 19 , further comprising the first polypeptide.