Method of purifying protein from inclusion bodies

Abstract

The present invention relates to processes for the purification of proteins. More specifically, methods for solubilizing and purifying proteins expressed in an insoluble form using low concentrations of chaotropic agents, such as guanidine salts, are provided. Also provided are methods for refolding proteins solubilized according to the present invention.

What is claimed is:  
     
         1 . A method for recovering a target protein from inclusion bodies, comprising: 
 (a) treating the inclusion bodies with a chaotropic agent at a concentration of about 0.7 to about 3.5M to solubilize the target protein; and    (b) recovering the target protein.    
     
     
         2 . The method according to  claim 1 , where said chaotropic agent is present at a concentration of about 1 to about 2 M.  
     
     
         3 . The method according to  claim 1 , wherein said chaotropic agent is selected from the group consisting of a guanidine salt and urea.  
     
     
         4 . The method according to  claim 3 , wherein said guanidine salt is selected from the group consisting of guanidine hydrochloride and guanidine isothiocyanate  
     
     
         5 . The method according to  claim 1 , wherein the inclusion bodies are obtained by lysing a cell selected from the group consisting of a bacterial microorganism, insect cells, mammalian cells and yeast cells.  
     
     
         6 . The method according to  claim 1 , where the target protein is a chemokine.  
     
     
         7 . The method according to  claim 1 , wherein, the target protein is refolded prior to recovering the target protein.  
     
     
         8 . The method according to  claim 1 , wherein, in step (b), recovery of the target protein includes subjecting the target protein to liquid chromatographic purification followed by tandem chromatography.  
     
     
         9 . The method according to  claim 1 , wherein, in step (b), recovery of the target protein includes subjecting the target protein to microfiltration followed by ultrafiltration.  
     
     
         10 . The method according to  claim 9  wherein, recovery of the target protein further includes refolding the target protein and subjecting the refolded target protein to liquid chromatographic purification.  
     
     
         11 . The target protein provided by the method according to  claim 1 .  
     
     
         12 . The target protein according to  claim 7 , selected from the group consisting of MPIF-1, MPEF-1d23, MIP-1α, M-CIF, MIP-4,Ck-β-13, Ck-α-4, and FGF-13.  
     
     
         13 . The target protein according to  claim 8 , selected from the group consisting of MPIF-1, MPIF-1d23, MIP-1α, M-CIF, MIP-4, Ck-β-13, Ck-α-4, and FGF-13.  
     
     
         14 . The target protein according to  claim 9 , selected from the group consisting of MPIF-1, MPIF-1d23, MIP-1α, M-CIF, MIP-4, Ck-β-13, Ck-α-4, and FGF-13.  
     
     
         15 . The method according to  claim 1 , where the recovered target protein is greater than 80% pure.  
     
     
         16 . The method according to  claim 8 , where the recovered target protein contains an endotoxin level of about 0.1 to about 1 EU/mg of protein.  
     
     
         17 . The method according to  claim 8 , wherein said recovered target protein includes refolded target protein.  
     
     
         18 . A method of recovering a secreted target protein comprising: 
 (a) subjecting the target protein to liquid chromatographic purification;    (b) subjecting the target protein of (a) to tandem chromatography; and    (c) recovering the target protein.    
     
     
         19 . The method according to  claim 18 , where the target protein is a chemokine.  
     
     
         20 . The purified target protein according to  claim 19 , selected from the group consisting of MPIF-1, MPIF-1d23, MIP-1α, M-CIF, MIP-4, Ck-β-13 Ck-α-4 and FGF-13.