Method for protein isolation in anoxic conditions
The present invention relates to a method for the isolation of proteins that comprise disulfide-bonds in their native conformation. Essentially, a method of the present makes the use of reducing agents such as β-mercaptoethanol or dithiothreitol in protein isolation methods obsolete. A method of the present invention is particularly suitable for the isolation of precursor proteins such as proinsulin from recombinant cells.
1 . A method of isolating proteins that comprise disulfide-bonds in their native conformation, said method comprising isolating said protein under essentially anoxic conditions.
2 . The method according to claim 1 , wherein said essentially anoxic conditions comprise an essentially anoxic atmosphere.
3 . The method according to claim 1 , wherein said protein is a recombinant hybrid protein present as inclusion bodies of recombinant cells.
4 . The method according to claim 3 , wherein said isolation comprises the harvesting and disruption of said cells, the isolation of said inclusion bodies and/or stabilization of the isolated product.
5 . The method according to claim 1 , wherein said protein is a precursor protein.
6 . The method according to claim 1 , wherein said protein is insulin.
7 . The method according to claim 2 , wherein said essentially anoxic atmosphere is a nitrogen atmosphere.
8 . The method according to claim 4 , wherein said recombinant cells are cells of E. coli strain Sφ733 carrying and expressing the plasmid pDBAST-RAT-N-7-1.