Purification of cystathionine beta-synthase

• US 9,243,239 B2
• Filed: 03/14/2013
• Issued: 01/26/2016
• Est. Priority Date: 03/26/2012
• Status: Active Grant

• Current Assignee: The Board of Regents of the University of Colorado (University of Colorado System)
• Original Assignee: The Board of Regents of the University of Colorado (University of Colorado System)
• Inventors: Kraus, Jan P., Majtan, Tomas, Naveh, David, Carrillo, Richard
• Primary Examiner(s): Yamasaki, Robert
• Assistant Examiner(s): Knight, Teresa E
• Application Number:
  US13/830,494
• Publication Number:
  US 20130251700A1
• Time in Patent Office: 1,048 Days
• US Class Current: 1/1
• CPC Class Codes: A61K 38/51 Lyases (4)
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: B01D 15/363 Anion-exchange
• CPC Class Codes: B01D 15/3823 of other types, e.g. avidin, streptavidin, biotin
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12Y 402/01022 Cystathionine beta-synthase (4.2.1.22)
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

This invention provides chromatographic methods for the purification of a cystathionine β-Synthase (CBS) protein, particularly truncated variants thereof and compositions and pharmaceutical compositions prepared therefrom.

Claims

1. A method for purifying cystathionine β

-synthase (CBS) protein, wherein the CBS protein is the CBS protein of SEQ ID NO;

02, or a truncation thereof, the method comprising the steps of;

(a) providing a CBS-containing solution comprising one or a plurality of impurities; and

(b) first performing chromatographic separation of the CBS-containing solution using an ion exchange chromatography column; and

(c) second performing a chromatographic separation using a metal affinity chromatography (IMAC) resin, wherein the impurities are removed thereby.

2. The method of claim 1, further comprising the step of performing chromatographic separation using a Hydrophobic Interaction Chromatography (HIC) column.

3. The method of claim 1, further comprising the step of performing chromatographic separation using a ceramic hydroxyapatite resin.

4. The method of claim 1, wherein the ion exchange chromatography column is a weak anion exchanger.

5. The method of claim 4, wherein the weak anion exchanger is a DEAE-Sepharose FF column.

6. The method of claim 1, wherein the metal affinity chromatography (IMAC) resin is charged with a divalent metal cation.

7. The method of claim 6, wherein the divalent metal cation is nickel, copper, cobalt or zinc.

8. The method of claim 7, wherein the divalent metal ion is zinc.

9. The method of claim 1, further comprising eluting CBS from the metal affinity chromatography (IMAC) resin with an elution buffer comprising imidazole.

10. The method of claim 1, wherein the truncated CBS protein has an amino acid sequence identified by SEQ ID NO:

3.

11. The method of claim 1, wherein the CBS-containing solution is a clarified CBS solution.

12. The method of claim 1, wherein the CBS is produced in a recombinant cell.

13. The method of claim 12, wherein the recombinant cell is a bacterial cell.

14. The method of claim 13, wherein the CBS-containing solution is obtained by homogenizing recombinant bacterial cells expressing a recombinant construct comprising a nucleic acid sequence encoding CBS.

15. The method of claim 14, wherein the CBS nucleic acid encodes a truncated CBS protein.

16. The method of claim 15, wherein the truncated CBS protein has been truncated to an ending position of one of amino acid residues from 382-532, 382-550 or 543-550 of SEQ ID NO:

2.

17. The method of claim 16, wherein the CBS nucleic acid sequence comprises SEQ ID NO. 4.

18. The method of claim 16, wherein the nucleic acid sequence encoding the truncated CBS protein is optimized for expression in E. coli cells.

19. The method of claim 12, wherein the recombinant cells are E. coli cells.