Recombinant Production of Heparin Binding Proteins

Abstract

A process for recovering and purifying refolded heparin binding proteins produced in heterologous host cells includes the step of incubation of the solubilized protein with a polyanionic species such as dextran sulfate.

1 . A process for recovering a heparin binding protein from a prokaryotic cell culture, the process comprising the steps of 
 (a) isolating said heparin binding protein from the periplasm of said prokaryotic cell culture;    (b) denaturing said isolated heparin binding protein in a first buffered solution comprising a chaotropic agent and a reducing agent;    (c) incubating said denatured heparin binding protein in a second buffered solution comprising a chaotropic agent and a sulfated polyanionic agent for such a time and under such conditions that refolding of the heparin binding protein occurs; and    (d) recovering said refolded heparin binding protein, wherein there is about a 2 to 5-fold increase in refolded heparin binding protein recovered compared to incubating with no sulfated polyanionic agent.    
   
   
       2 . The process of  claim 1 , wherein the heparin binding protein is a heparin binding growth factor.  
   
   
       3 . The process of  claim 2 , wherein the heparin binding growth factor is vascular endothelial growth factor (VEGF).  
   
   
       4 . The process of  claim 3 , wherein the VEGF is VEGF 165 .  
   
   
       5 . The process of  claim 1 , wherein the sulfated polyanionic agent is between about 3,000 daltons and 10,000 daltons.  
   
   
       6 . The process of  claim 3 , wherein said second buffered solution comprises a dextran sulfate.  
   
   
       7 . The process of  claim 3 , wherein said second buffered solution comprises sodium sulfate.  
   
   
       8 . The process of  claim 3 , wherein said second buffered solution comprises heparin.  
   
   
       9 . The process of  claim 6 , wherein the dextran sulfate is between about 8,000 and 10,000 daltons.  
   
   
       10 . The process of  claim 3 , wherein said first and second buffered solutions comprise HEPPS pH 8.0.  
   
   
       11 . The process of  claim 1 , wherein said second buffered solution further comprises a reducing agent.  
   
   
       12 . The process of  claim 11 , wherein the reducing agent of the second buffered solution comprises a combination of cysteine and DTT.  
   
   
       13 . The process of  claim 1 , wherein said second buffered solution further comprises a nonionic detergent.  
   
   
       14 . The process of  claim 1 , wherein said second buffered solution further comprises arginine and/or lysine.  
   
   
       15 . The process of  claim 1 , wherein said recovery step (d) comprises sequentially contacting said refolded heparin binding protein to a hydroxyapatite chromatographic support, a first hydrophobic interaction chromatographic support; a cationic chromatographic support, and a second hydrophobic interaction chromatographic support, and selectively eluting the heparin binding protein from each support.  
   
   
       16 . The process of  claim 15 , wherein said first and second hydrophobic interaction chromatographic support is selected from the group consisting of butyl-, propyl-, octyl- and aryl-agarose resins.  
   
   
       17 . The process of  claim 15 , wherein said first hydrophobic interaction chromatographic support is a butyl-agarose support and said second hydrophobic interaction chromatographic support is a phenyl-agarose support resin.  
   
   
       18 . The process of  claim 1 , wherein said recovery step (d) comprises sequentially contacting said refolded heparin binding protein to a cation exchange support; a hydrophobic interaction chromatographic support, and an ion exchange chromatographic support, and selectively eluting the heparin binding protein from each support.  
   
   
       19 . A method for recovering a heparin binding protein from a prokaryotic cell culture, the method comprising the steps of 
 (a) isolating said heparin binding protein from the periplasm of said prokaryotic cell culture;    (b) denaturing said isolated heparin binding protein in a first buffered solution comprising a chaotropic agent and a reducing agent;    (c) incubating said denatured heparin binding protein in a second buffered solution comprising a chaotropic agent and a sulfated polyanionic agent for such a time and under such conditions that refolding of the heparin binding protein occurs, wherein there is about a 2 to 5-fold increase in refolded heparin binding protein recovered compared to incubating with no sulfated polyanionic agent; and    (d) sequentially contacting said refolded heparin binding protein with a hydroxyapatite chromatographic support, a first hydrophobic interaction chromatographic support, a cationic chromatographic support, and a second hydrophobic interaction chromatographic support, and selectively eluting the heparin binding protein from each support.    
   
   
       20 . A method for purifying a heparin binding protein, the method comprising the steps of sequentially contacting a refolded heparin binding protein with a hydroxyapatite chromatographic support, a first hydrophobic interaction chromatographic support, a cationic chromatographic support, and a second hydrophobic interaction chromatographic support, and selectively eluting the heparin binding protein from each support.  
   
   
       21 . A method for recovering a heparin binding protein from a prokaryotic cell culture, the method comprising the steps of 
 (a) isolating said heparin binding protein from the periplasm of said prokaryotic cell culture;    (b) denaturing said isolated heparin binding protein in a first buffered solution comprising a chaotropic agent and a reducing agent;    (c) incubating said denatured heparin binding protein in a second buffered solution comprising a chaotropic agent and a sulfated polyanionic agent for such a time and under such is conditions that refolding of the heparin binding protein occurs, wherein there is about a 2 to 3-fold increase in refolded heparin binding protein recovered compared to incubating with no sulfated polyanionic agent; and,    (d) sequentially contacting said refolded heparin binding protein with a cation exchange support; a hydrophobic interaction chromatographic support, and an ion exchange chromatographic support, and selectively eluting the heparin binding protein from each support.    
   
   
       22 . The method of  claim 19  or  21 , wherein the polyanionic agent is between about 3,000 daltons and 10,000 daltons.  
   
   
       23 . A method for purifying a heparin binding protein, the method comprising the steps of sequentially contacting a refolded heparin binding protein with a cation exchange support; a hydrophobic interaction chromatographic support, and an ion exchange chromatographic support, and selectively eluting the heparin binding protein from each support.