Methods for refolding proteins containing free cysteine residues

Abstract

The present invention relates to novel methods for making and refolding insoluble or aggregated proteins having free cysteines in which a host cell expressing the protein is exposed to a cysteine blocking agent. The soluble, refolded proteins produced by the novel methods can then be modified to increase their effectiveness. Such modifications include attaching a PEG moiety to form PEGylated proteins.

1 . A method for preparing a refolded, soluble form of an insoluble or aggregated protein that is an anti-angiogenesis factor and which contains one or more added free cysteine residues, comprising the following steps, in order, of:
 a) causing a host cell to express a protein containing one or more added free cysteine residues that is an anti-angiogenesis factor in an insoluble or aggregated form;   b) lysing the cell by chemical, enzymatic or physical means;   c) separating soluble proteins from insoluble or aggregated proteins;   d) following step (c) and prior to step (e), dissolving the insoluble or aggregated protein from step (c) in a solution comprising a denaturing agent, a reducing agent and a cysteine blocking agent that forms a reversible mixed disulfide with cysteine, to solubilize and reduce the insoluble or aggregated protein, and to expose the insoluble or aggregated protein to the cysteine blocking agent; and   e) refolding the protein by reducing the concentrations of the denaturing agent and reducing agents to levels sufficient to allow the protein to renature into a soluble, biologically active form, wherein said refolding step occurs in the presence of the cysteine blocking agent, and wherein the cysteine blocking agent forms a reversible mixed disulfide with at least one added free cysteine residue in said protein.   
     
     
         2 . The method of  claim 1 , wherein said step (b) of lysing comprises lysing the host cell in the presence of the cysteine blocking agent used in step (d). 
     
     
         3 . The method of  claim 1 , wherein said step (b) of lysing comprises lysing the host cell in the presence of a denaturing agent. 
     
     
         4 . The method of  claim 1 , wherein said step (b) of lysing comprises lysing the host cell in the presence of a denaturing agent and the reducing agent used in step (d). 
     
     
         5 . The method of  claim 1 , wherein said cysteine blocking agent is selected from the group consisting of cysteine, cysteamine, reduced glutathione or thioglycolic acid. 
     
     
         6 . The method of  claim 1 , wherein said cysteine blocking agent is cysteine. 
     
     
         7 . The method of  claim 1 , wherein said reducing agent and said cysteine blocking agent of said step (d) are the same compound. 
     
     
         8 . The method of  claim 7 , wherein said cysteine blocking agent is selected from the group consisting of cysteine, cysteamine, reduced glutathione or thioglycolic acid. 
     
     
         9 . The method of  claim 1 , wherein said cysteine blocking agent of step (d) is a dithiol that, when reduced, acts as a cysteine blocking agent. 
     
     
         10 . The method of  claim 9 , wherein said dithiol is selected from the group consisting of cystine, cystamine, oxidized glutathione, or dithioglycolic acid. 
     
     
         11 . The method of  claim 1 , wherein the reducing agent is dithiothreitol (DTT) or 2-mercaptoethanol. 
     
     
         12 . The method of  claim 1 , wherein said denaturing agent is selected from the group consisting of: urea, guanidine and N-lauroyl sarcosine. 
     
     
         13 . The method of  claim 1 , wherein said step (e) of refolding comprises refolding the protein in the presence of glycerol. 
     
     
         14 . The method of  claim 1 , wherein said step (e) of refolding comprises refolding the protein in the presence of an oxidizing agent selected from the group consisting of oxygen, a dithiol, iodine, hydrogen peroxide, dihydroascorbic acid, tetrathionate, or O-iodosobenzoate. 
     
     
         15 . The method of  claim 1 , wherein step (e) of refolding comprises refolding the protein in the presence of a metal ion. 
     
     
         16 . The method of  claim 1 , wherein said step (e) of refolding comprises refolding the protein in the presence of a denaturing agent. 
     
     
         17 . The method of  claim 1 , wherein said step (e) of refolding comprises refolding the protein in the presence of a dithiol. 
     
     
         18 . The method of  claim 1 , wherein said step (e) of refolding occurs in the presence of a reducing agent. 
     
     
         19 . The method of  claim 1 , wherein said protein is a cysteine variant of a protein selected from the group consisting of endostatin, angiostatin, and the extracellular domain of a vascular endothelial growth factor (VEGF) receptor. 
     
     
         20 . The method of  claim 1 , further comprising attaching a cysteine-reactive moiety to at least one added free cysteine in said isolated protein to form a cysteine modified protein. 
     
     
         21 . The method of  claim 20 , wherein the cysteine-reactive moiety is selected from the group consisting of a polyethylene glycol, a polyvinyl pyrolidone, a carbohydrate, a dextran, a peptide, a lipid and a polysaccharide. 
     
     
         22 . The method of  claim 1 , further comprising the step of:
 f) isolating the refolded, soluble protein from step (e).   
     
     
         23 . The method of  claim 22 , further comprising the steps of:
 g) exposing the isolated protein to a disulfide-reducing agent; and   h) exposing the protein to a cysteine-reactive moiety to obtain a cysteine-modified protein, wherein the cysteine-reactive moiety is attached to at least one added cysteine in said isolated protein.   
     
     
         24 . The method of  claim 23 , wherein said cysteine-reactive moiety is selected from the group consisting of a polyethylene glycol, a polyvinyl pyrolidone, a dextran, a carbohydrate, a peptide, a lipid and a polysaccharide. 
     
     
         25 . A method for preparing and isolating a refolded, soluble form of an insoluble or aggregated protein that is an anti-angiogenesis factor and which contains one or more added free cysteine residues, comprising the steps of:
 a) causing a host cell to express a protein containing one or more added free cysteine residues that is an anti-angiogenesis factor in an insoluble or aggregated form;   b) lysing the cell by chemical, enzymatic or physical means in the presence of a denaturing agent, a reducing agent and a cysteine blocking agent, wherein the insoluble or aggregated protein is denatured and reduced, and is in the presence of the cysteine blocking agent prior to step (c);   c) refolding the protein by reducing the concentrations of the denaturing agent and reducing agents to levels sufficient to allow the protein to renature into a soluble, biologically active form, wherein said refolding step occurs in the presence of the cysteine blocking agent, and wherein the cysteine blocking agent forms a reversible mixed disulfide with at least one added free cysteine residue in said protein; and   d) isolating the refolded, soluble protein from step (c).