Fusion proteins
View Patent ↗The invention provides a single chain, polypeptide fusion protein, comprising: a non-cytotoxic protease, or a fragment thereof, which protease or protease fragment is capable of cleaving a protein of the exocytic fusion apparatus of a target cell; a Targeting Moiety that is capable of binding to a Binding Site on the target cell, which Binding Site is capable of undergoing endocytosis to be incorporated into an endosome within the target cell; a protease cleavage site at which site the fusion protein is cleavable by a protease, wherein the protease cleavage site is located between the non-cytotoxic protease or fragment thereof and the Targeting Moiety; and a translocation domain that is capable of translocating the protease or protease fragment from within an endosome, across the endosomal membrane and into the cytosol of the target cell.
1. A single chain, polypeptide fusion protein having an N-terminus and a C-terminus, said fusion protein comprising:
(a) an L-chain of a botulinum neurotoxin;
(b) a PAR1 ligand located C-terminal of the L-chain;
(c) a protease cleavage site at which site the fusion protein is cleavable by a protease, wherein the protease cleavage site is located between the L-chain and the PAR1 ligand; and
(d) a translocation domain that comprises the H N domain of a botulinum neurotoxin;
wherein the PAR1 ligand is located between the protease cleavage site and the translocation domain; and
wherein the fusion protein lacks the natural binding function of the Hc component of a clostridial neurotoxin H-chain.
2. The fusion protein according to claim 1 , wherein the fusion protein comprises a purification tag.
3. The fusion protein according to claim 2 , wherein the purification tag is joined to the fusion protein by a peptide spacer molecule.
4. The fusion protein according to claim 1 , wherein the translocation domain is separated from the PAR1 ligand by a peptide spacer molecule.
5. The fusion protein according to claim 1 , wherein the protease cleavage site is a non-clostridial protease cleavage site selected from the group consisting of: DDDDK (SEQ ID NO: 49); IEGR (SEQ ID NO: 50); IDGR (SEQ ID NO: 51); ENLYFQG (SEQ ID NO: 52); LVPRGS (SEQ ID NO: 53); and LEVLFQGP (SEQ ID NO: 54).
6. A nucleic acid sequence encoding the polypeptide fusion protein according to claim 1 .
7. A DNA vector, comprising a promoter, a nucleic acid sequence according to claim 6 located downstream of the promoter, and a terminator located downstream of said nucleic acid sequence.