Recombinant Production of Heparin Binding Proteins
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.