Patent ID: 7658933

Claim:
A method of preparing an activated di-chain fusion protein, said di-chain fusion protein comprising a first domain comprising a botulinum neurotoxin light chain, a second domain comprising a botulinum neurotoxin heavy chain H N translocation domain, and a third domain comprising an amino acid sequence of SEQ ID NO:14, wherein the first domain and the second domain are linked by a disulfide bond, said method comprising: a) expressing a single-chain fusion protein from a nucleic acid sequence that encodes a single-chain fusion protein, wherein said single-chain fusion protein comprises: i) a first domain comprising a botulinum neurotoxin light chain, ii) a second domain comprising a botulinum neurotoxin heavy chain H N translocation domain, iii) an enterokinase cleavage site located between the first domain and the second domain, and iv) a third domain comprising the amino acid sequence set forth in SEQ ID NO:14, wherein the first domain and the second domain are linked by a disulfide bond; b) contacting the single-chain fusion protein of a) with an enterokinase protease under conditions permitting cleavage of the enterokinase cleavage site; and c) isolating activated di-chain fusion protein from b) having the enterokinase cleavage site cleaved, thereby preparing an activated di-chain fusion protein.