Universal procedure for refolding recombinant proteins

Abstract

A universal folding method that has been demonstrated to be effective in refolding a variety of very different proteins expressed in bacteria as inclusion bodies has been developed. Representative proteins that can be dissolved and refolded in biologically active form, with the native structure, are shown in Table I. The method has two key steps to unfold and then refold the proteins expressed in the inclusion bodies. The first step is to raise the pH of the protein solution in the presence of denaturing agents to pH greater than 9, preferably 10. The protein solution may be maintained at the elevated pH for a period of up to about 24 hours, or the pH immediately decreased slowly, in increments of about 0.2 pH units/24 hours, until the solution reaches a pH of about 8.0, or both steps used. In the preferred embodiment, purified inclusion bodies are dissolved in 8 M urea, 0.1 M Tris, 1 mM glycine, 1 mM EDTA, 10 mM beta-mercaptoethanol, 10 mM dithiothreitol (DTT), 1 mM redued glutathion (GSH), 0.1 mM oxidized glutathion (GSSG), pH 10. The absorbance at 280 nm (OD280) of the protein solution is 5.0. This solution is rapidly diluted into 20 volumes of 20 mM Tris base. The resulting solutin is adjusted to pH 9.0 with 1 M HCl and is kept at 4° C. for 24 hr. The pH is adjusted to pH 8.8 and the solution is kept at 4° C. for another 24 hrs. This process is repeated until the pH is adjusted to 8.0. After 24 hr at pH 8.0, the refolded proteins can be concentrated by ultrafiltration and applied to a gel filtration column for purification.

1 . A method for refolding of recombinant proteins comprising 
 maintaining the protein at a pH of 9.0 or greater, in the presence of one or more chaotrophic and reducing agents, and    decreasing the pH of the solution gradually over a period of at least about 24 hrs to pH 8.0 to induce renaturation of at least a portion of the protein so that it qualitatively exhibits a biological activity and structure characteristic of the protein.    
   
   
       2 . The method of  claim 1  wherein pH is decreased in increments equivalent to 0.2 pH units per 24 hours.  
   
   
       3 . The method of  claim 1  wherein the protein is maintained at a pH of greater than 9.0 for a period of at least 24 hours.  
   
   
       4 . The method of  claim 1  wherein the pH is decreased by addition of acid.  
   
   
       5 . The method of  claim 1  wherein the pH is decreased by dilution or dialysis into a solution of a lower pH.  
   
   
       6 . The method of  claim 1  wherein the chaotrophic and denaturing reagents are selected from the group consisting of between 0.5 and 1.0 M urea, 0.1 mM to 100 mM beta-mercaptoethanol, 0.1 mM to 100 mM DTT, 0.1 mM to 10 mM reduced glutathion, and 0.1 mM to 10 mM oxidized glutathion.  
   
   
       7 . The method of  claim 1  wherein the protein is first extracted from bacterial inclusion bodies.  
   
   
       8 . The method of  claim 4  wherein the bacteria is  E. coli.    
   
   
       9 . The method of  claim 7  wherein the inclusion bodies are dissolved at a final pH of between greater than 9 and 10.  
   
   
       10 . The method of  claim 1 , wherein the protein is dissolved at a pH above about 10.0.  
   
   
       11 . The method of  claim 1 , wherein the protein is dissolved at a pH above about 11.0.  
   
   
       12 . The method of  claim 1 , wherein the protein is dissolved at a pH above about 12.0.  
   
   
       13 . The method of  claim 1  wherein the pH is decreased over a period of at least about 36 hours.  
   
   
       14 . The method of  claim 1 , wherein the pH of the solution is reduced more quickly at the higher pH and more gradually nearer the physiological pH range of the protein.  
   
   
       15 . The method of  claim 1  comprising the additional step of separating protein species which exhibits biological activity from inactive, or wrongly folded, protein species.