Method for protein isolation in anoxic conditions

Abstract

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.