Purification of cystathionine beta-synthase

Abstract

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

Claims

1. A method for purifying cystathionine -synthase (CBS) protein comprising: (a) providing a CBS-containing solution comprising one or a plurality of impurities; and (b) performing chromatographic separation of the CBS-containing solution using an ion exchange chromatography column; (c) performing chromatographic separation of the CBS-containing solution using a metal affinity chromatography (IMAC) resin; (d) performing chromatographic separation of the CBS-containing solution using a ceramic hydroxyapatite resin; and (e) performing chromatographic separation of the CBS-containing solution using a hydrophobic interaction chromatography (HIC) column; wherein the CBS protein comprises the amino acid sequence of SEQ ID NO: 2 or a truncation thereof and does not comprise a purification tag.

2. The method of claim 1, wherein the ion exchange chromatography column is a DEAE-Sepharose FF column, a DEAE-Sephacel column, a DEAE-cellulose column, a DEAE-Sephadex column, or a QAE-Sephadex column.

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

4. The method of claim 3, wherein the divalent metal ion is nickel, copper, cobalt, or zinc.

5. The method of claim 3, wherein the first chromatographic separation step is IMAC chromatography and the IMAC resin is charged with nickel or copper.

6. The method of claim 3, wherein the first chromatographic separation step is ion exchange chromatography followed by IMAC chromatography, and wherein the IMAC resin is charged with zinc.

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

8. The method of claim 1, wherein the CBS protein is a chemically cleaved or genetically engineered carboxyl terminal truncated CBS protein having a carboxyl-terminal truncation resulting in an ending position of one of amino acid residues 382-550 of the amino acid sequence of SEQ ID NO: 2.

9. The method of claim 1, wherein the CBS protein comprises the amino acid sequence of SEQ ID NO: 3.

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

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

12. The method of claim 11, wherein the CBS-containing solution is obtained by homogenizing recombinant cells expressing a recombinant construct comprising a nucleic acid sequence encoding the CBS protein.

13. The method of claim 12, wherein homogenizing is performed by disrupting the cells using sonication.

14. The method of claim 12, wherein homogenizing is performed by disrupting the cells by exposing the cells to a lysozyme.

15. The method of claim 12, wherein the nucleic acid sequence encoding the CBS protein comprises the nucleotide sequence of SEQ ID NO: 4.

16. The method of claim 1, wherein the CBS protein binds to a chromatography matrix during chromatographic separation.

17. The method of claim 16, further comprising eluting the CBS protein from the chromatography matrix.

18. A method of preparing a pharmaceutical composition comprising combining a purified CBS solution prepared according to the method of claim 1 and a pharmaceutically acceptable carrier.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) The following detailed description of the embodiments of the present invention can be best understood when read in conjunction with the following drawings.

(2) FIG. 1 is a purification train summary from scale-up generation runs using a multi-step chromatography method including DEAE-Sepharose-FF, Zn-IMAC and HIC chromatography.

(3) FIG. 2 is a purification summary from purification experiments using a DEAE-Sepharose-FF column and CBS purified using the non-optimized bacterial expression construct. Mobile phases included 10% ethylene glycol in addition to other components as set forth in the Examples.

(4) FIG. 3 is a purification train summary from scale-up generation runs using a multi-step chromatography method including DEAE-Sepharose-FF, Zn-IMAC, ceramic hydroxyapatite resin and HIC chromatography.

(5) FIG. 4 is a photoimage of a SDS page gel showing the relative amounts of CBS protein and impurities for each stage of the purification step using a DEAE column.

(6) FIG. 5 is a photoimage of a SDS page gel showing the relative amounts of CBS protein and impurities for a 3 column purification method including: a DEAE column, a Zn-IMAC column and HIC column.

(7) FIG. 6 is a photoimage of a SDS page gel showing the relative amounts of CBS protein and impurities for a 4 column purification method including: a DEAE column, a Zn-IMAC column, a ceramic hydroxyapatite resin and a HIC column.

(8) FIG. 7 is chromatograms demonstrating the components of the separated mixture following purification using Zn-IMAC.

(9) FIG. 8 is a purification summary from development runs using a Ni-IMAC column.

(10) FIG. 9 is a summary table demonstrating the total protein following a purification method using a Ni-IMAC column.

(11) FIG. 10 is a photoimage of a SDS page gel showing the relative amounts of CBS protein and impurities for each stage of the purification step using a Ni-IMAC column.

(12) FIG. 11 is a purification summary from scale-up generation runs using a Cu-IMAC column.

(13) FIG. 12 is a summary table demonstrating the total protein following a purification method using a Zn-IMAC column.

(14) FIG. 13 is a photoimage of a SDS page gel showing the relative amounts of CBS protein and impurities for each stage of the purification step using a Zn-IMAC column.

(15) FIG. 14 is a scheme of the purification method using multi-step chromatography purification steps.

DETAILED DESCRIPTION OF THE INVENTION

(16) This invention provides methods for purification of CBS protein, wherein said CBS protein is a naturally occurring truncated variant, or a chemically cleaved or genetically engineered truncate thereof, and particularly a truncated protein CBS produced in recombinant cells. In particular, the invention provides methods for the purification of a CBS protein that include the steps (a) providing a CBS-containing solution in the presence of at least one impurity; and (b) performing chromatographic separation of said CBS-containing solution using a metal affinity chromatography (IMAC) resin. In additional particular embodiments, the method comprises the steps of: (a) providing a CBS-containing solution in the presence of at least one impurity; and (b) performing chromatographic separation of said CBS-containing solution using an ion exchange chromatography column and a metal affinity chromatography (IMAC) resin.

(17) A particular chromatographic separation step in the certain embodiments of the methods provided by this invention comprises an ion exchange chromatography column. In one embodiment, the ion exchange chromatography column is an anion exchanger, preferably a weak anion exchanger. Various types of anion exchange resins can be used, including DEAE-Sephadex, QAE-Sephadex, DEAE-Sephacel, DEAE-cellulose and DEAE-Sepharose-FF. According to one embodiment, the anion exchange resin is DEAE-Sepharose-FF.

(18) Another particular chromatographic separation step in the certain of the methods provided by this invention comprises a metal affinity chromatography (IMAC) resin having appropriate pH and conductivity such to allow the protein to bind to the column while selective intermediate washes are used to remove weaker binding proteins and other molecular species. In certain embodiments, varying concentrations of imidazole were used to modulate the partitioning during the chromatography. Suitable metal affinity resins include immobilized metal affinity columns charged with a divalent metal ion including nickel, copper, cobalt or zinc. In certain embodiments of the methods of the invention, the metal affinity chromatography (IMAC) column is used following ion exchange chromatography. In such embodiments, the IMAC column is preferably charge with zinc as a divalent cation. In other embodiments of the inventive methods, the IMAC column is used as an initial chromatographic step. In such embodiments, nickel or copper divalent cations are preferably used to charge the IMAC column.

(19) Additional chromatographic steps provided in certain embodiments of the methods of this invention for purifying CBS from a CBS-containing solution include without limitation hydrophobic interaction chromatography (HIC). HIC is useful for removing impurities that have relatively closely related chromatographic properties that are eluted together with the target protein during the capture step.

(20) Further additional chromatographic steps provided in certain embodiments of the methods of this invention for purifying CBS from a CBS-containing solution include without limitation a ceramic hydroxyapatite resin. Ceramic hydroxyapatite or CHAP refers to an insoluble hydroxylated calcium phosphate of the formula (Ca.sub.10(PO.sub.4).sub.6(OH).sub.2), which has been sintered at high temperatures into a spherical, macroporous ceramic form. The methods of the invention also can be used with hydroxyapatite resin that is loose or packed in a column. The choice of column dimensions can be determined by the skilled artisan.

(21) Chromatography matrices useful in the method of the invention are materials capable of binding biochemical compounds, preferably proteins, nucleic acids, and/or endotoxins, wherein the affinity of said biochemical compounds to said chromatography matrix is influenced by the ion composition of the surrounding solution (buffer). Controlling the ion composition of said solution allows to use the chromatography materials of the invention either in subtractive mode (CBS passes through said chromatography matrix, at least certain contaminants bind to said chromatography matrix) or, preferably, in adsorptive mode (CBS binds to the chromatography matrix).

(22) In particular embodiments, the method for purification comprises the step of homogenizing host cells, particularly recombinant cells and in certain embodiments, recombinant cells producing mammalian, preferable human, CBS protein, wherein said recombinant construct encodes a CBS protein that is a naturally occurring truncated variant, or a genetically engineered truncate thereof, and particularly wherein said construct has been optimized for recombinant cell expression. In particular embodiments, said recombinant cells are microbial cells and particularly bacterial cells. In certain particular embodiments, the bacterial cells are E. coli cells and the CBS sequence has been engineered in the recombinant expression construct to be optimized for expression in said cells; a specific embodiment of such a nucleic acid sequence optimized for CBS expression in E. coli is set forth in SEQ ID NO: 4. In said methods, cells are harvested, e.g. by centrifugation, and optionally stored at 80 degree C. Homogenization of host cells is performed by disrupting the cells host using physical, chemical or enzymatic means or by a combination thereof. Advantageously, for purification from bacterial sources homogenation is performed by disrupting the cell wall of said bacterial host by sonication. Alternatively or additionally homogenizing is performed by destabilizing the bacterial cell wall of the host by exposure to a cell wall degrading enzyme such as lysozyme.

(23) The methods of the invention can further comprise a clarified CBS homogenate, wherein cell debris is removed from the homogenate by either filtration or centrifugation. In certain embodiments, clarifying is performed by centrifuging the homogenate at an effective rotational speed. The required centrifugation time depends inter alia on the volume of the homogenate, which can be determined empirically to obtain a sufficiently solid pellet. To obtain an essentially cell debris-free clarified homogenate a combination of centrifugation and filtration can be performed on the homogenate.

(24) The term recombinant cell as used herein refers to suitable cells (including progeny of such cells) from any species into which has been introduced a recombinant expression construct capable of expressing a nucleic acid encoding CBS protein, preferably human CBS protein and most particularly a human CBS protein that is a naturally occurring truncated variant, or a chemically cleaved or genetically engineered truncate thereof. In specific embodiments, the truncated CBS protein encoded by said recombinant expression construct has an amino acid sequence as set forth in SEQ ID NO: 3.

(25) The term, bacterial cell, as used herein refers to bacteria that produces a mammalian, preferably human, CBS protein inter alia using recombinant genetic methods including progeny of said recombinant cell, wherein said CBS protein is a naturally occurring truncated variant, or a genetically engineered truncate thereof.

(26) The term recombinant expression construct as used herein refers to a nucleic acid having a nucleotide sequence of a mammalian, preferably human, CBS protein, and sequences sufficient to direct the synthesis of CBS protein in cultures of cells into which the recombinant expression construct is introduced and the progeny thereof.

(27) As used herein, reference to CBS protein or polypeptide preferably includes a naturally occurring truncated variant, or a chemically cleaved or genetically engineered truncate thereof, or fusion proteins, or any homologue (variant, mutant) thereof, and specifically mammalian CBS and preferably human CBS. Such a CBS protein can include, but is not limited to, purified CBS protein, recombinantly produced CBS protein, soluble CBS protein, insoluble CBS protein, and isolated CBS protein associated with other proteins. In addition, a human CBS protein refers to a CBS protein from a human (Homo sapiens) preferably includes a naturally occurring truncated variant, or a chemically cleaved or genetically engineered truncate thereof. As such, a human CBS protein can include purified, partially purified, recombinant, mutated/modified and synthetic proteins. As disclosed herein and in related U.S. Pat. Nos. 8,007,787 and 7,485,307, the CBS protein truncates are advantageously soluble CBS proteins that are produced in bacteria without the creation of insoluble inclusion bodies.

(28) As used herein, the term homologue (or variant or mutant) is used to refer to a protein or peptide which differs from a naturally occurring protein or peptide (i.e., the prototype or wild-type protein) by modifications to the naturally occurring protein or peptide, but which maintains the basic protein and side chain structure of the naturally occurring form. Such changes include, but are not limited to: changes in one, few, or even several amino acid side chains; changes in one, few or several amino acids, including deletions (e.g., a truncated version of the protein or peptide), insertions and/or substitutions; changes in stereochemistry of one or a few atoms; and/or minor derivatizations, including but not limited to: methylation, glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation and/or addition of glycosylphosphatidyl inositol. A homologue can have enhanced, decreased, changed, or substantially similar properties as compared to the naturally occurring protein or peptide. A homologue can include an agonist of a protein or an antagonist of a protein.

(29) Homologues can be the result of natural allelic variation or natural mutation. A naturally occurring allelic variant of a nucleic acid encoding a protein is a gene that occurs at essentially the same locus (or loci) in the genome as the gene which encodes such protein, but which, due to natural variations caused by, for example, mutation or recombination, has a similar but not identical sequence. Allelic variants typically encode proteins having similar activity to that of the protein encoded by the gene to which they are being compared. One class of allelic variants can encode the same protein but have different nucleic acid sequences due to the degeneracy of the genetic code. Allelic variants can also comprise alterations in the 5 or 3 untranslated regions of the gene (e.g., in regulatory control regions). Allelic variants are well known to those skilled in the art.

(30) Homologues can be produced using techniques known in the art for the production of proteins including, but not limited to, direct modifications to the isolated, naturally occurring protein, direct protein synthesis, or modifications to the nucleic acid sequence encoding the protein using, for example, classic or recombinant DNA techniques to effect random or targeted mutagenesis. CBS variants are described in U.S. Pat. No. 8,007,787, which is incorporated herein by reference in its entirety; in particular and preferred embodiments, the reagents and methods of the invention set forth herein preferably include a naturally occurring truncated variant, or a chemically cleaved or genetically engineered truncate of human CBS protein. Particular truncated forms of SEQ ID NO: 3 according to the present invention include N-terminal deletion variants, C-terminal deletion variants, and variants having both N-terminal and C-terminal deletions.

(31) As used herein, substantially pure refers to a purity that allows for the effective use of the protein in vitro, ex vivo or in vivo. For a protein to be useful in vitro, ex vivo or in vivo, it is preferably substantially free of contaminants, other proteins and/or chemicals that might interfere or that would interfere with its use, or that at least would be undesirable for inclusion with a CBS protein (including homologues thereof).

(32) As used herein an enriched CBS solution is a solution subjected to one or more purification steps.

(33) The purity of protein can be determined by calculating fold purification, i.e. a formula that provides a measure of how much more a purified solution is compared to a less purified solution or crude extract. Fold purification is calculated using the following formula:

(34) Specific Activity Final Fraction/Specific Activity Crude Fraction

(35) Another measurement to assess purity is the specific activity which measures the purity of an enzyme. Specific activity can be measured using the following formula:

(36) $\frac{\mathrm{Units}}{\mathrm{mL}}\frac{\mathrm{mL}}{\mathrm{mg}}=\frac{\mathrm{Units}}{\mathrm{mg}}$

(37) CBS protein compositions provided by this invention are useful for regulating biological processes and particularly, processes associated with the catalysis of the pyridoxal 5-phosphate (PLP)-dependent condensation of serine and homocysteine to form cystathionine. In particular, compositions of the present invention are useful for producing cystathionine and cysteine in vitro or for treating a patient that will benefit from increased CBS activity (e.g., a patient with homocystinuria). In certain embodiments, the invention provides said compositions of CBS protein, preferably human CBS protein, wherein said CBS protein is a naturally occurring truncated variant, or a chemically cleaved or genetically engineered truncate of human CBS protein, as pharmaceutical compositions comprising said CBS protein and a pharmaceutically acceptable carrier.

(38) As used herein, a pharmaceutically acceptable carrier includes pharmaceutically acceptable excipients and/or pharmaceutically acceptable delivery vehicles, suitable for use in suitable administration of the composition in vitro, ex vivo or in vivo. Suitable in vitro, in vivo or ex vivo administration preferably comprises any site where it is desirable to regulate CBS activity. Suitable pharmaceutically acceptable carriers are capable of maintaining a CBS protein as provided by this invention in a form that, upon arrival of the protein at the target cell or tissue in a culture or in patient, the protein has its expected or desired biological activity. Examples of pharmaceutically acceptable carriers include, but are not limited to water, phosphate buffered saline, Ringer's solution, dextrose solution, serum-containing solutions, Hank's solution, other aqueous physiologically balanced solutions, oils, esters and glycols. Aqueous carriers can contain suitable auxiliary substances required to approximate the physiological conditions of the recipient, for example, by enhancing chemical stability and isotonicity. Compositions of the present invention can be sterilized by conventional methods and/or lyophilized.

(39) Each reference described and/or cited herein is incorporated by reference in its entirety.

(40) The following examples are provided for the purpose of illustration and are not intended to limit the scope of the present invention.

EXAMPLES

Example 1. Production of Truncated CBS Protein in Bacteria

(41) A truncated human CBS variant lacking specific portions of the non-conserved regions (r-hCSC; SEQ ID No: 3) were constructed and over-expressed using the previously described E. coli based expression system (Kozich and Kraus, 1992, supra). In the modification of this system disclosed herein (i.e., expressing the truncate rather than the full-length CBS protein), the CBS truncate encoded by SEQ ID NO: 3 was expressed without any fusion partner under the control of the tac promoter. Constructs encoding the truncated human CBS protein variant r-hCSC (SEQ ID NO: 4) were generated by a modification of the previously described pHCS3 CBS expression construct (Kozich and Kraus, 1992, Hum. Mutat. 1, 113-123) which contains the CBS full-length coding sequence (SEQ ID NO: 1) cloned into pKK388.1. In this construct, CBS expression was governed by the IPTG inducible lac promoter. To generate C-terminal deletion constructs, CBS cDNA fragments spanning the desired nucleotide residues were amplified using primers incorporating Sph I and Kpn I sites to the 5 and 3 respective ends of the PCR product. All PCR products were then cut with Sph I and Kpn I and cloned by ligation into the pHCS3 vector digested with Sph I and Kpn I. An Sph I site naturally occurs in the CBS cDNA, just upstream of the antisense primer hybridization site (base pare position 1012, according to the CBS cDNA numbering, ref. 25). PCR products thus generated were then digested with Nco I and Sph I and ligated into the pHCS3 plasmid cut with the same enzymes.

(42) TABLE-US-00001 pKKCBS414-551 sense: (SEQIDNO:5) CGTAGAATTCACCTTTGCCCGCATGCTGAT(SphI) antisense: (SEQIDNO:6) TACGGGTACCTCAACGGAGGTGCCACCACCAGGGC(KpnI)

(43) Finally, the construct was transformed into E. coli BL21 (Stratagene). The authenticity of the construct was verified by DNA sequencing using a Thermo Sequenase Cy5.5 sequencing kit (Amersham Pharmacia Biotech) and the Visible Genetics Long-Read Tower System-V3.1 DNA sequencer according to the manufacturer's instructions.

(44) Bacterial Expression Analysis of CBS Deletion Mutants and Growth of E. coli

(45) BL21 cells bearing the CBS truncation mutant construct, induction of expression and the generation of crude cell lysates were performed as described previously (Maclean et al., 2002, Hum. Mutat. 19(6), 641-55). Briefly, bacteria were grown at 37 C. aerobically in 1 L NZCYMT media (Gibco/BRL, Gaithersburg, Md.) containing 75 g/mL ampicillin and 0.001% thiamine in the presence or absence of 0.3 mM -aminolevulinate (-ALA) until they reached turbidity of 0.5 at 600 nm. IPTG was then added to 0.5 mM and the bacteria were grown further. The insoluble fraction was prepared as follows: after the centrifugation of the sonicated homogenate, pelleted cell debris were thoroughly washed with chilled Ix Tris-buffered saline, pH 8.0. The pellets were then resuspended in 1 ml of the lysis buffer (Maclean et al., ibid.) followed by a brief sonication in order to homogenize the insoluble fraction.

(46) CBS Activity Assay

(47) CBS activity was determined by a previously described radioisotope assay using [.sup.14C] serine as the labeled substrate (Kraus, 1987, Methods Enzymol. 143, 388-394). Protein concentrations were determined by the Lowry procedure (Lowry et al., 1951, J. Biol. Chem. 193, 265-275) using bovine serum albumin (BSA) as a standard. One unit of activity is defined as the amount of CBS that catalyzes the formation of 1 mol of cystathionine in 1 h at 37 C.

(48) Denaturing and Native Polyacrylamide Gel Electrophoresis and Western BlottingWestern blot analysis of crude cell lysates under both denaturing and native conditions was performed as described previously (Janosik, 2001, supra) with some modifications. Soluble fractions of E. coli lysates containing the expressed mutant protein were mixed with sample buffer and run on a 6% native PAGE without a stacking gel. The final composition of the sample buffer was: 50 mM Tris-HCl, pH 8.9, 1 mM DTT, 10% glycerol, 0.001% bromophenol blue. Detection of heme was performed using a previously described method that relies on heme peroxidase activity (Vargas et al., 1993, Anal. Biochem. 209(2), 323-6).

(49) Densitometric Scanning Analysis

(50) Quantitative densitometry analysis was performed using the Imagemaster ID (version 2.0) software (Pharmacia). To construct a calibration curve, 50, 75, 100, 250, 500 and 1000 ng of purified wild type CBS protein were run on an SDS-PAGE together with crude cell lysates of the individual mutants. Following electrophoresis, Western blot immunoanalysis was conducted using rabbit anti-CBS serum. The signals corresponding to the experimentally observed CBS mutant subunits were all within the linear range of the calibration curve constructed with purified human CBS.

Example 2. Preparation of Crude Extraction

(51) Crude CBS protein-containing extracts was prepared for use in downstream chromatography steps. Frozen pellets (cells) obtained from fermentation of recombinant bacteria producing human truncated CBS variant (r-hCSC; SEQ ID No: 3) were lysed, wherein said bacteria expressed truncated human CBS encoded by SEQ ID NO: 4. Lysis buffer for initial isolations contained 1 mM DTT, 1% Triton X-100, and Protease Inhibitor. These components were eventually removed from the buffer. The buffer used for the final isolations that produced material for scale-up runs consisted of 20 mM Sodium Phosphate, 50 mM NaCl, 0.1 mM PLP (pH 7.2), with lysozyme added to a concentration of 2 mg/mL after homogenization. Following mixing with lysozyme for 1 hr at 4 C., the homogenate was sonicated until viscosity was reduced and then subjected to centrifugation at 20,000 rpm (48,000g) for 30 min. The supernatant was collected, aliquoted, and stored at 70 C. until use. Generally, the crude extract was thawed at 37 C. prior to chromatographic purification.

Example 3. DEAE-Sepharose FF Chromatography

(52) DEAE-Sepharose FF was used in this Example of the purification methods for CBS because it possesses good capacity and flow properties and has been manufactured consistently for several years. This step employed a drip/gravity column that contained approximately 6 mL of resin. The column was equilibrated in Sodium Phosphate buffer with 50 mM NaCl, pH 7.0. Loading of the crude extract was targeted at approximately 20 mg total protein/mL resin. After loading the column, the red color of the load was concentrated near the top of the column. Following a wash with equilibration buffer, the column was washed with a buffer containing 150 mm NaCl, whereby the majority of color eluted from the column (all steps were performed at pH 7.0). Essentially all color was removed from the column with a 300 mM NaCl wash. Based on these results, a column was packed that could be operated in flow mode. The conditions employed equilibration/loading at a NaCl concentration 50 mM, with elution at 250 mM NaCl. The final conditions required dilution of the column load with H.sub.2O to approach the ionic strength of the equilibration/wash buffer (50 mM NaCl), and elution with 137 mM NaCl (FIGS. 1, 2 and 3). Samples were analyzed by SDS-PAGE to determine the relative amounts of CBS protein and impurities (FIG. 4). The following tables represent column operational parameters and data from the scale-up runs that employed them.

(53) TABLE-US-00002 TABLE 1 Operational Parameters for DEAE Capture Step Column Contact time Column load target NaCl Concentration Volumes Column vol./flow rate Process Step (total protein mg/mL) (with 20 mM Na.sub.3PO.sub.4 pH 7.0) (mL) (min.) Equilibration N/A 50 mM 3-5 10 Load 20-25 mg/mL Approx. 50 mM Variable 15 Wash N/A 50 mM 3 10 Elution N/A 137 mM Variable* 15 2M NaCl Strip N/A 2M 3 10

(54) Note:

(55) Eluate collection started at approximately 0.4 AU and ended at approximately 0.55 AU. Void volume was typically approximately 1 column volume.

(56) TABLE-US-00003 TABLE 2 Data from Scale-up Runs (n = 6) Input Column loading (per mL Resin) Output Total Protein Recovery Fold Purif. (mg) Units (%) (By S.A.) 14.5-19.8 3275-5443 79.3-93.0 2.5-3.3 Range 18.2 4451 86 2.8 Average

Example 4. IMAC Chromatography

(57) The ability for an immobilized metal affinity column (IMAC) to separate CBS protein from impurities and other contaminants from a biological source, such as a recombinant bacterial cell homogenate, was demonstrated. Because of the desire to avoid low pH conditions (<5, anecdotal), varying concentrations of imidazole were used to modulate partitioning during the chromatography.

(58) Copper (Cu) was tested as a candidate species of IMAC column based on its relatively strong binding characteristics. Prior to being applied to the IMAC column, the CBS solution was adjusted to 0.4M NaCl. The results indicated that capture was near complete, with an acceptable activity recovery (70-80%). Recovery of CBS was obtained using 100 mM imidazole, which resulted in significant precipitation upon thawing from storage at 70 C. (FIG. 11). In addition, there was only a small increase in purity relative to the load. Thus, experiments employing NiIMAC were conducted as the metal of choice. In these experiments, the CBS sample was run through a G-25 column to remove dithiothreitol (DTT) prior to loading the solution onto the IMAC column. Purity enhancement remained low and selectivity was similar to Cu.sup.++, as evidenced by a relatively small A.sub.280 peak in the high imidazole strip fraction. (FIGS. 8, 9 and 10).

(59) The relatively weak binding Zn was also tested. Although capture, wash and elution conditions required significantly lower imidazole concentrations, potential for purity enhancement provided positive results due to the significant size of the A.sub.280 peaks in the post-load wash and high imidazole strip fractions. 0.4 M NaCl and 0.01% Triton X-100 were added to the equilibration and wash buffers to minimize non-specific binding. (FIGS. 1 and 3). Samples were analyzed by SDS-PAGE to determine the relative amounts of CBS protein and impurities (FIG. 13). The results of the IMAC experiments are presented in FIG. 7. The following tables represent column operational parameters and data from the scale-up runs that employed them.

(60) TABLE-US-00004 TABLE 3 Operational Parameters for Zn-IMAC Step Contact time Process Column load target Imidazole Concentration Column Volumes Column vol./flow rate Step (total protein mg/mL) (with 20 mM Na.sub.3PO.sub.4 pH 7.0) (mL) (min.) Equilibration N/A 1 mM 3 10 Load 10 0 Variable 10 Wash N/A 1 mM 3 10 Elution N/A 11 mM Variable* 10 Strip N/A 100 mM 3 10

(61) Note:

(62) Eluate collection started at approximately 0.25 AU and ended at approximately 0.16 AU. Void volume was typically approximately 1.5 column volumes.

(63) TABLE-US-00005 TABLE 4 Data from Scale-up Runs (n = 5) Input Column loading (per mL Resin) Output Total Protein Recovery Fold Purif. (mg) Units (%) (By S.A.) 6.5-9.3 4414-7038 71.8-84.6 1.3-1.6 Range 8.1 5687 80 1.4 Average

Example 5. HIC Chromatography

(64) Multiple experiments were conducted to identify the parameters for HIC chromatography. Initial drip column experiments were conducted that employed a resin with a relatively strong binding ligand (phenyl) with an IMAC eluate as starting material/load. This experiment resulted in empirically complete binding at 1.3M (NH.sub.4).sub.2SO.sub.4. However, there was evidence of significant retention of CBS on the column even after washing with a low ionic strength buffer. Based on these results, a resin with a weaker binding ligand (butyl) was tested. Initial experiments with this resin showed no apparent capture at 0.5M (NH.sub.4).sub.2SO.sub.4. The non-binding flow through of this column experiment was collected and adjusted to 1.25M (NH.sub.4).sub.2SO.sub.4, and reloaded on to a column equilibrated to the same concentration of (NH.sub.4).sub.2SO.sub.4. In this case there was evidence of significant binding to the column. A 20 column volume (NH.sub.4).sub.2SO.sub.4 gradient elution was performed from 1.25M to 0.25M (NH.sub.4).sub.2SO.sub.4 with fractions collected. SDS-PAGE analysis of the fractions indicated that there was significant potential for impurity clearance on the lower end of the gradient. Experiments utilizing step gradient washes at varying concentrations of (NH.sub.4).sub.2SO.sub.4 determined the final operational parameters. (FIGS. 1 and 3). Those parameters and the scale-up run data are summarized in the tables below.

(65) TABLE-US-00006 TABLE 5 Operational parameters for HIC Step (n = 6) Column Contact time Process Column load target (NH.sub.4).sub.2SO.sub.4 Concentration Volumes Column vol./flow rate Step (total protein mg/mL) (with 20 mM Na.sub.3PO.sub.4 pH 7.0) (mL) (min.) Equilibration N/A 1.4M 3 10 Load 5-10 1.4M Variable 10 Wash N/A 1.4M 3 10 Elution N/A 1.1M Variable* 10 Strip N/A 0.05M NaCl 3 10

(66) Note:

(67) Eluate collection starts at approx. 0.25 AU and ends at approx. 0.15 AU. Void volume typically approx. 1.4 column volumes.

(68) TABLE-US-00007 TABLE 6 Data from Scale-up Runs (n = 5) Input Column loading (per mL Resin) Output Total Protein Recovery Fold Purif. (mg) Units (%) (By S.A.) 5.1-7.2 5375-9248 77.8-92.7 1.0-1.3 Range 6.3 7638 85 1.2 Average

Example 6. CHAP Chromatography

(69) Ceramic hydroxyapatite is a resin that has a unique, potentially mixed binding mode chemistry that was utilized in a CBS purification method. CBS displayed acidic characteristics and therefore initial investigation focused on using phosphate-modulated partitioning. The initial experiments utilized HIC eluate that was buffer exchanged into a 0.05M NaCl, 0.005M Potassium Phosphate (pH 6.8) buffer. A 5 mL ceramic hydroxyapatite (Type 1) cartridge was equilibrated in the same buffer and the conditioned HIC eluate was loaded onto the column. There was no visible breakthrough of protein (as measured by A.sub.280) during the load and subsequent wash with equilibration/wash buffer. A linear gradient (5%) of 0.005M to 0.5M Potassium Phosphate was then run and fractions were collected. Based on the chromatogram, samples were analyzed by SDS-PAGE to determine the relative amounts of CBS protein and impurities. In subsequent experiments (based on analysis of the results of previous experiments), step washes with varying levels of phosphate were employed to determine optimal conditions for load, wash, and elution steps. In addition, the composition of buffer salts was transitioned from Potassium to Sodium Phosphate. (FIG. 3). The following tables represent column operational parameters and data from the scale-up runs that employed them.

(70) TABLE-US-00008 TABLE 7 Operational Parameters for CHAP Step Contact time Column load target Na.sub.3PO.sub.4 Concentration Column Volumes Column vol./flow rate Process Step (total protein mg/mL) (with 50 mM NaCl, pH 7.0) (mL) (min.) Equilibration N/A 10 mM 3 6 Load 10-15 10 mM Variable 6 Wash N/A 30 mM 3 6 Elution N/A 90 mM Variable* 6 Strip N/A 150 mM 3 6

(71) Note: Eluate collection started at approximately 0.20 AU and ends at approximately 0.16 AU. Void volume was typically approximately 1.0 column volumes.

(72) TABLE-US-00009 TABLE 8 Data from Scale-up Runs (n = 5) Input Column loading (per mL Resin) Output Total Protein Recovery Fold Purif. (mg) Units (%) (By S.A.) 9.9-12.2 11205-12297 84.6-92.4 1.1-1.2 Range 11.1 11751 89 1.2 Average

Example 7. Integrated Process Results

(73) The particular multi-step method described in these Examples was evaluated at the scale of a 60 mL capture column. All of the purification trains utilized starting material (crude extract) obtained from fermentations that were seeded with recombinant cells comprising a construct comprising a truncated variant of human CBS encoded by a nucleic acid having codons optimized for expression in E. coli. This construct resulted in starting material that was approximately 2-fold higher in specific activity, and significantly impacted the final purity achieved from the integrated purification method. The overall purification results using the multi-step method were measured by SDS-PAGE and Specific Activity (FIGS. 5 and 6). The results demonstrated that the purity and specific activity met or exceeded that of the purified tagged truncated CBS. All Specific Activities of final column eluates obtained by the largest scale currently possible exceeded 1200 U/mg total protein. The following table summarizes the overall purification results from the scale-up runs.

(74) TABLE-US-00010 TABLE 9 Overall Results from Scale-Up Runs Total Recovery (%) Fold Purification Range Average Range Average 3 Column Train (n = 3) 57-60 58 5.7-6.2 5.9 4 Column Train (n = 2) 47-52 50 4.6-5.4 5.0

(75) Specific Activity of Final Column Eluate=1206-1509.

(76) Having described the invention in detail and by reference to specific embodiments thereof, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims. More specifically, although some aspects of the present invention are identified herein as particularly advantageous, it is contemplated that the present invention is not necessarily limited to these particular aspects of the invention.

(77) TABLE-US-00011 TABLE10 CSSequences Molecule SEQIDNO Sequence NativehumanCS 1 atgccttctgagaccccccaggcagaagtggggcccacaggctgccccca nucleicacidsequence ccgctcagggccacactcggcgaaggggagcctggagaaggggtccccag aggataaggaagccaaggagcccctgtggatccggcccgatgctccgagc aggtgcacctggcagctgggccggcctgcctccgagtccccacatcacca cactgccccggcaaaatctccaaaaatcttgccagatattctgaagaaaa tcggggacacccctatggtcagaatcaacaagattgggaagaagttcggc ctgaagtgtgagctcttggccaagtgtgagttcttcaacgcgggcgggag cgtgaaggaccgcatcagcctgcggatgattgaggatgctgagcgcgacg ggacgctgaagcccggggacacgattatcgagccgacatccgggaacacc gggatcgggctggccctggctgcggcagtgaggggctatcgctgcatcat cgtgatgccagagaagatgagctccgagaaggtggacgtgctgcgggcac tgggggctgagattgtgaggacgcccaccaatgccaggttcgactccccg gagtcacacgtgggggtggcctggcggctgaagaacgaaatccccaattc tcacatcctagaccagtaccgcaacgccagcaaccccctggctcactacg acaccaccgctgatgagatcctgcagcagtgtgatgggaagctggacatg ctggtggcttcagtgggcacgggcggcaccatcacgggcattgccaggaa gctgaaggagaagtgtcctggatgcaggatcattggggtggatcccgaag ggtccatcctcgcagagccggaggagctgaaccagacggagcagacaacc tacgaggtggaagggatcggctacgacttcatccccacggtgctggacag gacggtggtggacaagtggttcaagagcaacgatgaggaggcgttcacct ttgcccgcatgctgatcgcgcaagaggggctgctgtgcggtggcagtgct ggcagcacggtggcggtggccgtgaaggctgcgcaggagctgcaggaggg ccagcgctgcgtggtcattctgcccgactcagtgcggaactacatgacca agttcctgagcgacaggtggatgctgcagaagggctttctgaaggaggag gacctcacggagaagaagccctggtggtggcacctccgtgttcaggagct gggcctgtcagccccgctgaccgtgctcccgaccatcacctgtgggcaca ccatcgagatcctccgggagaagggcttcgaccaggcgcccgtggtggat gaggcgggggtaatcctgggaatggtgacgcttgggaacatgctctcgtc cctgcttgccgggaaggtgcagccgtcagaccaagttggcaaagtcatct acaagcagttcaaacagatccgcctcacggacacgctgggcaggctctcg cacatcctggagatggaccacttcgccctggtggtgcacgagcagatcca gtaccacagcaccgggaagtccagtcagcggcagatggtgttcggggtgg tcaccgccattgacttgctgaacttcgtggccgcccaggagcgggaccag aagtga NativehumanCS 2 MPSETPQAEVGPTGCPHRSGPHSAKGSLEKGSPEDKEAKEPLWIRPDAPS polypeptidesequence RCTWQLGRPASESPHHHTAPAKSPKILPDILKKIGDTPMVRINKIGKKFG LKCELLAKCEFFNAGGSVKDRISLRMIEDAERDGTLKPGDTIIEPTSGNT GIGLALAAAVRGYRCIIVMPEKMSSEKVDVLRALGAEIVRTPTNAREDSP ESHVGVAWRLKNEIPNSHILDQYRNASNPLAHYDTTADEILQQCDGKLDM LVASVGTGGTITGIARKLKEKCPGCRIIGVDPEGSILAEPEELNQTEQTT YEVEGIGYDFIPTVLDRIVVDKWEKSNDEEAFTFARMLIAQEGLLCGGSA GSTVAVAVKAAQELQEGQRCVVILPDSVRNYMTKFLSDRWMLQKGFLKEE DLTEKKPWWWHLRVQELGLSAPLTVLPTITCGHTIEILREKGFDQAPVVD EAGVILGMVTLGNMLSSLLAGKVQPSDQVGKVIYKQFKQIRLTDTLGRLS HILEMDHFALVVHEQIQYHSTGKSSQRQMVEGVVTAIDLLNEVAAQERDQ K Truncated,Human 3 MPSETPQAEVGPTGCPHRSGPHSAKGSLEKGSPEDKEAKE CSpolypeptide PLWIRPDAPSRCTWQLGRPASESPHHHTAPAKSPKILPDI sequence LKKIGDTPMVRINKIGKKFGLKCELLAKCEFFNAGGSVKD RISLRMIEDAERDGTLKPGDTIIEPTSGNTGIGLALAAAV RGYRCIIVMPEKMSSEKVDVLRALGAEIVRTPTNARFDSP ESHVGVAWRLKNEIPNSHILDQYRNASNPLAHYDTTADEI LQQCDGKLDMLVASVGTGGTITGIARKLKEKCPGCRIIGV DPEGSILAEPEELNQTEQTTYEVEGIGYDFIPTVLDRTVV DKWFKSNDEEAFTFARMLIAQEGLLCGGSAGSTVAVAVKA AQELQEGQRCVVILPDSVRNYMTKFLSDRWMLQKGFLKEE DLTEKKPWWWHLR Truncated,Optimized 4 ATGCCGTCAGAAACCCCGCAGGCAGAAGTGGGTCCGACGG HumanCSnucleic GTTGCCCGCACCGTAGCGGTCCGCATTCTGCAAAAGGCAG acidsequence TCTGGAAAAAGGTTCCCCGGAAGATAAAGAAGCCAAAGAA CCGCTGTGGATTCGTCCGGACGCACCGTCACGCTGTACCT GGCAGCTGGGTCGTCCGGCAAGCGAATCTCCGCATCACCA TACGGCTCCGGCGAAAAGTCCGAAAATTCTGCCGGATATC CTGAAGAAAATTGGTGACACCCCGATGGTTCGTATCAACA AAATCGGCAAAAAATTCGGTCTGAAATGCGAACTGCTGGC TAAATGTGAATTTTTCAATGCGGGCGGTTCCGTGAAAGAT CGTATCTCACTGCGCATGATTGAAGATGCTGAACGCGACG GCACCCTGAAACCGGGTGATACGATTATCGAACCGACCTC TGGCAACACGGGTATCGGTCTGGCACTGGCGGCGGCAGTC CGTGGTTATCGCTGCATTATCGTGATGCCGGAAAAAATGA GCTCTGAAAAAGTTGATGTCCTGCGTGCTCTGGGCGCGGA AATTGTTCGTACCCCGACGAATGCCCGCTTCGACAGTCCG GAATCCCATGTGGGTGTTGCATGGCGCCTGAAAAACGAAA TCCCGAATTCGCACATTCTGGATCAGTATCGTAACGCTAG CAATCCGCTGGCGCATTACGATACCACGGCCGACGAAATC CTGCAGCAATGTGATGGCAAACTGGACATGCTGGTCGCTT CTGTGGGTACCGGCGGTACCATTACGGGCATCGCGCGTAA ACTGAAAGAAAAATGCCCGGGCTGTCGCATTATCGGTGTG GATCCGGAAGGCAGTATTCTGGCGGAACCGGAAGAACTGA ACCAGACCGAACAAACCACGTATGAAGTTGAAGGCATCGG TTACGATTTTATTCCGACCGTCCTGGATCGCACGGTGGTT GACAAATGGTTCAAAAGCAATGACGAAGAAGCCTTTACCT TCGCACGTATGCTGATCGCTCAGGAAGGTCTGCTGTGCGG TGGTTCAGCAGGTTCGACGGTCGCAGTGGCAGTTAAAGCT GCGCAGGAACTGCAAGAAGGTCAACGTTGTGTCGTGATTC TGCCGGATTCTGTTCGCAACTACATGACCAAATTTCTGAG TGACCGTTGGATGCTGCAAAAAGGCTTCCTGAAAGAAGAA GATCTGACCGAGAAAAAACCGTGGTGGTGGCACCTGCGCT AA