Purification of cystathionine beta-synthase

• US 10,160,962 B2
• Filed: 12/16/2015
• Issued: 12/25/2018
• 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: Carrillo, Richard, Kraus, Jan P., Majtan, Tomas, Naveh, David
• Primary Examiner(s): Holland, Paul J
• Application Number:
  US14/970,814
• Publication Number:
  US 20160177287A1
• Time in Patent Office: 1,105 Days
• 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 producing an enriched cystathionine β

-synthase (CBS) protein-containing solution produced by a method comprising the steps of;

(a) providing a CBS protein-containing solution comprising one or more impurities, wherein the CBS protein does not include a purification tag;

(b) performing chromatographic separation of the CBS protein-containing solution using an immobilized metal affinity chromatography (IMAC) resin charged with a divalent metal ion, wherein the one or more impurities are removed; and

(c) performing at least one additional purification step selected from the group consisting of;

hydrophobic interaction chromatography (HIC), ceramic hydroxyapatite (CHAP), and ion exchange chromatography,

wherein the CBS protein of the CBS protein-containing solution comprises a chemically cleaved or genetically engineered carboxyl terminal truncated CBS protein derived from the amino acid sequence of SEQ ID NO;

2.

2. The method of claim 1, wherein the divalent metal ion is nickel, copper, cobalt, or zinc.

3. The method of claim 2, wherein the divalent metal ion is zinc.

4. The method of claim 1, wherein the chemically cleaved or genetically engineered carboxyl truncated CBS protein comprises a carboxyl-terminal truncation spanning any of amino acid residues 383-532, 383-551, or 543-551 of the amino acid sequence of SEQ ID NO:

2.

5. The method of claim 1, wherein the chemically cleaved or genetically engineered truncated CBS protein comprises the amino acid sequence of SEQ ID NO:

3.

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

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

8. The method of claim 7, wherein the recombinant cell is a bacterial cell.

9. The method of claim 8, wherein the bacterial cell is an E. coli cell.

10. The method of claim 8, wherein the CBS protein-containing solution is obtained by homogenizing recombinant bacterial cells expressing a recombinant construct comprising a nucleic acid sequence encoding the CBS protein.

11. The method of claim 10, wherein the nucleic acid sequence encoding the CBS protein comprises the nucleotide sequence of SEQ ID NO:

4.

12. The method of claim 10, wherein the nucleic acid sequence encoding the CBS protein is optimized for expression in E. coli cells.