Manufacture of di-chain recombinant
Methods for producing di-chain BoNT/A from single chain BoNT/A using Lys-C followed by purification using a hydrophobic surface.
1. A method for producing soluble di-chain BoNT/A protein, said method comprising:
a) providing a soluble single-chain BoNT/A protein;
b) contacting said BoNT/A protein with endoproteinase Lys-C (Lys-C) in solution; and
c) separating the soluble BoNT/A protein from the Lys-C by contacting the solution containing soluble BoNT/A protein and Lys-C with a hydrophobic surface, wherein the soluble BoNT/A protein binds with preference to the hydrophobic surface.
2. The method according to claim 1 , wherein the soluble single-chain BoNT/A protein is produced (i) in a host cell, by expressing a nucleic acid encoding said single-chain BoNT/A protein in an expression system; or (ii) in an in vitro expression system.
3. The method according to claim 2 , wherein the host cell is a bacterial host cell and the expression system is a bacterial expression system.
4. The method according to claim 3 , wherein the bacterial expression system is an E. coli expression system and the host cell is an E. coli cell.
5. The method according to claim 2 , wherein said soluble single-chain BoNT/A protein is expressed in the cytoplasm of said host cell.
6. The method according to claim 2 , wherein said soluble single-chain BoNT/A protein is expressed at a level of at least 5 mg/L.
7. The method according to claim 2 , comprising lysis of the bacterial or E. coli host cell to provide a bacterial or E. coli host cell homogenate containing said soluble single-chain BoNT/A protein.
8. The method according to claim 1 , wherein the hydrophobic surface is an inert matrix to which a ligand consisting of aryl or alkyl groups is attached.
9. The method according to claim 8 , wherein the ligand is selected from the group consisting of: butyl, phenyl and octyl ligands.
10. The method according to claim 9 , wherein a high performance hydrophobic surface is used.