Non-cytotoxic protein conjugates
View Patent ↗The present invention is directed to non-cytotoxic protein conjugates for inhibition or reduction of exocytic fusion in a nociceptive sensory afferent cell. The protein conjugates comprise: (i) a Targeting Moiety (TM), wherein the TM is an agonist of a receptor present on a nociceptive sensory afferent cell, and wherein the receptor undergoes endocytosis to be incorporated into an endosome within the nociceptive sensory afferent cell; (ii) a non-cytotoxic protease or a fragment thereof, wherein the protease or protease fragment is capable of cleaving a protein of the exocytic fusion apparatus of the nociceptive sensory afferent cell; and (iii) a Translocation Domain, wherein the Translocation Domain translocates the protease or protease fragment from within the endosome, across the endosomal membrane, and into the cytosol of the nociceptive sensory afferent cell. Nucleic acid sequences encoding the protein conjugates, methods of preparing same and uses thereof are also described.
1. 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.
2. The method according to claim 1 , wherein the enterokinase cleavage site has the amino acid sequence DDDDK (SEQ ID NO:102).
3. The method according to claim 1 , wherein the nucleic acid sequence is encompassed within an expression vector.
4. An activated di-chain fusion protein produced by the method according to claim 1 .