Methods of refolding proteins

Abstract

Methods are disclosed for refolding proteins of the TGF-β family of proteins. The methods employ as refolding agents one or more compounds which are non-detergent zwitterionic compounds, such as sulfobetaines, substituted pyridines, substituted pyrroles and acid substituted aminocyclohexanes.

We claim:  
     
         1 . A method of expressing a protein from the transforming growth factor-beta [TGF-β] superfamily comprising: 
 a) culturing prokaryotic cells which have been transformed with a DNA encoding a TGF-β protein under conditions suitable for the production of recombinant TGF-β protein; and  
 b) refolding the recombinantly produced TGF-β protein in refolding media comprising a non-detergent zwitterionic compound.  
 
     
     
         2 . The method of  claim 1 , wherein the prokaryotic cells are  E. coli.    
     
     
         3 . The method of  claim 2  wherein the protein is a bone morphogenetic protein.  
     
     
         4 . The method of  claim 3 , wherein the non-detergent zwitterionic compound is a substituted non-detergent sulfobetaine.  
     
     
         5 . The method of  claim 4 , wherein the non-detergent zwitterionic compound is 3-(1-pyridinio)-1-propanesulfonate.  
     
     
         6 . The method of  claim 1 , wherein the non-detergent zwitterionic compound is a compound with a nitrogen-containing aromatic ring or a nitiogen-containing aliphatic ring where nitrogen is in the form of a quaternary amine, said compound further containing an electron accepting substituent group.  
     
     
         7 . The method of  claim 6 , wherein the non-detergent zwitterionic compound is selected from the group consisting of N-methyl-N-piperidine propane sulfonic acid, trigonelline hydrochloride, and 1-carboxymethyl pyridinium chloride.  
     
     
         8 . A method of expressing a protein from the transforming growth factor-beta [TGF-β] superfamily comprising: 
 a) culturing prokaryotic cells which have been transformed with a DNA encoding a TGF-β protein under conditions suitable for the production of recombinant TGF-β protein; and  
 b) refolding the recombinantly produced TGF-β protein in refolding media comprising an acid or amide substituted pyridine compound.  
 
     
     
         9 . The method of  claim 8 , wherein the substitute p,ridine compound is selected from the group consisting of pyridine 3-sulfonic acid, pyridine-2 carboxylic acid, picolinic acid, 3-pyridylacetic acid hydrochloride, 4-pyridylacetic acid hydrochloride, 2-pyridine ethane sulfonic acid, 3-pyridylhydroxymethane sulfonic acid, nicotinic acid, isonicotinic acid and nicotinamide.  
     
     
         10 . The method of  claim 9 , wherein the prokaryotic cells are bacterial cells.  
     
     
         11 . The method of  claim 10 , wherein the bacterial cells are  E. coli.    
     
     
         12 . The method of  claim 9  wherein the protein is a bone morphogenetic protein.  
     
     
         13 . A method of expressing a protein from the transforming growth factor-beta [TGF-β] superfamily comprising: 
 a) culturing prokaryotic cells which have been transformed with a DNA encoding a TGF-β protein under conditions suitable for the production of recombinant TGF-β protein; and  
 b) refolding the recombinantly produced TGF-β protein in refolding media comprising an acid or amide substituted pyrrole compound.  
 
     
     
         14 . The method of  claim 13 , wherein the substituted pyrrole compound is selected from the group consisting of pyrrole 3-sulfonic acid, pyrrole-2 carboxylic acid, 3-pyrrole acetic acid hydrochloride, 2-pyrrole acetic acid hydrochloride 2-pyrrole ethane sulfonic acid, 3-pyrrolehydroxymethane sulfonic acid.  
     
     
         15 . The method of  claim 14 , wherein the prokaryotic cells are bacterial cells.  
     
     
         16 . The method of  claim 15 , wherein the bacterial cells are  E. coli.    
     
     
         17 . The method of  claim 14  wherein the protein is a bone morphogenetic protein.  
     
     
         18 . A method of expressing a protein from the transforming growth factor-beta [TGF-β] superfamily comprising: 
 a) culturing prokaryotic cells which have been transformed with a DNA encoding a TGF-β protein under conditions suitable for the production of recombinant TGF-β protein; and  
 b) refolding the recombinantly produced TGF-β protein in refolding media comprising an acid substituted aminohexane compound.  
 
     
     
         19 . The method of  claim 18 , wherein the acid substituted aminohexane compound is selected from the group consisting of 2-aminohexanecarboxylic acid, CHES, CAPS and CAPSO.  
     
     
         20 . The method of  claim 19 , wherein the prokaryotic cells are bacterial cells.  
     
     
         21 . The method of  claim 20 , wherein the bacterial cells are  E. coli.    
     
     
         22 . The method of  claim 19  wherein the protein is a bone morphogenetic protein.  
     
     
         23 . The method of  claim 1 , wherein the non-detergent zwitterionic compound contains a hydrophobic group selected from the group consisting of nitrogen-containing aromatic rings and quaternary nitrogen-containing aliphatic rings, and an electrophilic end group selected from the group consisting of acid and amide, and wherein the the electrophilic end group is no more than four carbons removed from the nitrogen-containing ring.