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 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.