Purification of cystathionine beta-synthase

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.

1 . A composition comprising a purified cystathionine β-synthase (CBS) protein produced by a method comprising the steps of:
 (a) providing a CBS protein-containing solution, said solution comprising one or more impurities; 
 (b) first performing chromatographic separation of the CBS protein-containing solution of (a) using an ion exchange chromatography column; and 
 (c) second performing a chromatographic separation using a metal affinity chromatography (IMAC) resin, wherein the one or more impurities are removed from the CBS protein-containing solution of (a), 
 
       wherein the CBS protein of the CBS protein-containing solution of (a) comprises a chemically cleaved or genetically engineered carboxyl-terminal truncated CBS protein derived from SEQ ID NO: 2. 
     
     
         2 . The composition of  claim 1 , wherein the method further comprises at least one additional step of performing chromatographic separation using a ceramic hydroxyapatite (CHAP) resin or a Hydrophobic Interaction Chromatography (HIC). 
     
     
         3 . The composition of  claim 1 , wherein the metal affinity chromatography (IMAC) resin is charged with a divalent metal cation. 
     
     
         4 . The composition of  claim 1 , wherein the chemically cleaved or genetically engineered truncated CBS protein has an amino acid sequence identified by SEQ ID NO: 3. 
     
     
         5 . The composition of  claim 1 , wherein the ion exchange chromatography column is a weak anion exchanger. 
     
     
         6 . The composition of  claim 5  wherein the weak anion exchanger is selected from the group consisting of: a DEAE-Sepharose FF column, a DEAE-Sephacel column, a DEAE-cellulose column, a DEAE-Sephadex column, and a QAE-Sephadex column. 
     
     
         7 . The composition of  claim 1 , wherein the metal affinity chromatography (IMAC) resin is charged with a divalent metal cation. 
     
     
         8 . The composition of  claim 7 , wherein the divalent metal cation is nickel, copper, cobalt, or zinc. 
     
     
         9 . The composition of  claim 8 , wherein the divalent metal ion is zinc. 
     
     
         10 . The composition of  claim 1 , wherein the method further comprises eluting the CBS protein from the metal affinity chromatography (IMAC) resin with an elution buffer comprising imidazole. 
     
     
         11 . The composition of  claim 1 , wherein the purified CBS protein has an amino acid sequence identified by SEQ ID NO: 3. 
     
     
         12 . The composition of  claim 1 , wherein the CBS protein-containing solution is a clarified CBS solution. 
     
     
         13 . The composition of  claim 1 , wherein the CBS protein is produced in a recombinant cell. 
     
     
         14 . The composition of  claim 13 , wherein the recombinant cell is a bacterial cell. 
     
     
         15 . The composition of  claim 14 , wherein the recombinant cell is an  E. coli  cell. 
     
     
         16 . The composition of  claim 1 , wherein the CBS-protein containing solution is obtained by homogenizing recombinant bacterial cells expressing a recombinant construct comprising a nucleic acid sequence encoding CBS. 
     
     
         17 . The composition of  claim 16 , wherein the recombinant bacterial cells are  E. coli  cells. 
     
     
         18 . The composition of  claim 16 , wherein the nucleic acid sequence encodes a naturally occurring or a genetically engineered truncated CBS protein. 
     
     
         19 . The composition of  claim 18 , wherein the amino acid sequence of the CBS protein has an ending position of one of amino acid residues from 382-532, 382-550, or 543-550 compared to SEQ ID NO: 2. 
     
     
         20 . The composition of  claim 18 , wherein the nucleic acid sequence comprises SEQ ID NO. 4. 
     
     
         21 . The composition of  claim 18 , wherein the nucleic acid sequence encoding the truncated CBS protein is optimized for expression in  E. coli  cells. 
     
     
         22 . The composition of  claim 1 , wherein the CBS protein does not include a tag. 
     
     
         23 . The composition of  claim 1 , wherein the CBS protein is eluted during chromatographic separation. 
     
     
         24 . The composition of  claim 1 , wherein the CBS protein binds to a chromatography matrix during chromatographic separation. 
     
     
         25 . A pharmaceutical composition comprising the composition of  claim 1  and a pharmaceutically acceptable carrier.