TH74141B - Recombinant production of heparin-binding protein - Google Patents

Abstract

DC60 (11/04/50) A process for the separation and purification of heparin-binding proteins at A new folded and manufactured hetero locust cell. Including the process of curing protein Then dissolved with poly anionic substances such as Dextran sulfate A process for the separation and purification of proteins binded to heparin at A new folded and manufactured hetero locust cell. Including the process of curing protein Then dissolved with poly anionic substances such as Dextran sulfate

Claims (23)

Revised August 5, 2019, Page 1 of 3. Claims 1. A process for the recovery of heparin-binding protein from cultured prokaryote cells. The heparin-binding protein is a heparin-binding vascular endothelial growth factor (VEGF). The process consists of the following steps: (a) isolation of the heparin-binding protein from the periplasm of the cultured prokaryote cells; (b) denaturation of the heparin-binding protein in a first buffer solution containing a chaotropic agent and a reducing agent; (c) incubation of the heparin-binding protein in a second buffer solution containing a chaotropic agent and a sulfated polyanionic agent for a period of time and in (d) the conditions under which heparin-binding protein refolding occurs, and (d) the recovery of such refolded heparin-binding protein, which resulted in a 2 to 5 fold increase in recoverable refolded heparin-binding protein compared to incubation without sulfated polyanionic agents. 1. The process for isolating heparin-binding proteins from cultured prokaryote cells consists of the following steps: (a) isolation of the heparin-binding proteins from the periplasm of the cultured prokaryote cells; (b) denaturing the isolated heparin-binding proteins in a first buffer solution containing a dissociating agent and a reducing agent; (c) incubating the denatured heparin-binding proteins in a second buffer solution containing a dissociating agent and a sulfated polyanionic agent for a specified time and under conditions in which heparin-binding protein refolding occurs; and (d) isolation of the refolded heparin-binding proteins, resulting in a 2 to 5-fold increase in the isolated refolded heparin-binding proteins compared to incubation without the sulfated polyanionic agent. 2. The process of claim 1, where the hepparin-binding protein is the hepparin-binding growth factor. 3. The process of claim 2, where the heparin-binding growth factor is endothelial growth factor (VEGF). 4. The process of the third claim, in which VEGF is VEGF165. 5. The process of claim 1, in which sulfated polyanionic compounds have a molecular weight between approximately 3,000 and 10,000 Daltons. 6. The process of claim 3, in which the second buffer solution contains dextran sulfate. 7. The process of claim 3, in which the second buffer solution contains sodium sulfate. 8. The process of claim 3, in which the second buffer solution contains heparin. 9. The process of claim number 6, in which dextran sulfate has a molecular weight between approximately 8,000 and 10,000 daltons. 10. The process of claim 3, in which the first and second buffer solutions contain HEPPS pH 8.0. 11. The process of claim 1, in which the second buffer solution contains an additional reducing agent. 12. The process of claim 1, in which the second buffer solution contains an additional component consisting of cysteine and DTT together. 13. The process of claim 1, in which the second buffer solution contains an additional component consisting of a non-charged detergent. 14. The process of claim 1, in which the second buffer solution contains an additional component consisting of arginine and/or lysine. 15. The process of claim 1, in which the separation step (d) is performed, involves sequential contact between the newly folded heparin-binding protein and the hydroxyapatite chromatographic support, the first hydrophobic interaction chromatographic support, the cationic exchange chromatographic support, and the second hydrophobic interaction chromatographic support, and the selective elution of the heparin-binding protein from each support. 16. The process of claim 15, in which the first hydrophobic interaction chromatographic support is selected from a group consisting of butyl-, propyl-, otyl-, and aryl-agarose resins. 17. The process of claim 15, whereby the first hydrophobic interaction chromatography support is a butyl-agarose resin support and the second hydrophobic interaction chromatography support is a phenyl-agarose resin support. 18. The process of claim 1, in which the separation step (d) is performed, consists of sequential contact between the newly folded heparin-binding protein and the cationic exchange support, the hydrophobic interaction chromatographic support, and the ion-exchange chromatographic support, and the selective removal of the heparin-binding protein from each support. 19. Method for isolating heparin-binding proteins from cultured prokaryotic cells. The method consists of the following steps: (a) isolation of the heparin-binding proteins from the periplasm of the cultured prokaryotic cells; (b) denaturing the isolated heparin-binding proteins in a first buffer solution containing a dissociating agent and a reducing agent; (c) incubating the denatured heparin-binding proteins in a second buffer solution containing a dissociating agent and a sulfated polyanionic agent for a specified time and under conditions in which heparin-binding protein refolding occurs, resulting in a 2 to 5-fold increase in the isolated refolded heparin-binding proteins compared to incubation without the sulfated polyanionic agent; and (d) sequential contact between the refolded heparin-binding proteins. This was done with hydroxyapatite chromatographic supports, primary hydrophobic chromatographic support, cationic exchange chromatographic support, and secondary hydrophobic chromatographic support, and selective elution of heparin-binding proteins from each support. 20. Method for purifying heparin-binding proteins. The method consists of sequential contact steps between the newly folded heparin-binding proteins and the hydroxyapatite chromatographic support, the first hydrophobic interaction chromatographic support, the cationic exchange chromatographic support, and the second hydrophobic interaction chromatographic support, followed by selective elution of the heparin-binding proteins from each support. 21. Method for isolating heparin-binding proteins from cultured prokaryote cells. The method consists of the following steps: (a) isolation of the heparin-binding proteins from the periplasm of the cultured prokaryote cells; (b) denaturing the isolated heparin-binding proteins in a first buffer solution containing a dissociating agent and a reducing agent; (c) incubating the denatured heparin-binding proteins in a second buffer solution containing a dissociating agent and a sulfated polyanionic agent for a specified time and under conditions in which heparin-binding protein refolding occurs, resulting in a 2 to 3 fold increase in the isolated refolded heparin-binding proteins compared to incubation without the sulfated polyanionic agent; and (d) contact, respectively, between the refolded heparin-binding proteins. This was done with cationic exchange chromatography (CEC) supports; hydrophobic interaction chromatography (HIIC) supports; and ion-exchange chromatography (ION) supports, and selective elution of heparin-binding proteins from each support. 22. Method of claim 19 or 21, in which polyanionic substances have a molecular weight between approximately 3,000 Daltons and 10,000 Daltons. 23. Method for purifying heparin-binding proteins. The method consists of sequential contact steps between the newly folded heparin-binding protein and a cation-exchange chromatography support; a hydrophobic interaction chromatography support and an ion-exchange chromatography support; and selective elution of the heparin-binding protein from each support.