Patent Application
20160177287
The present invention generally relates to methods for purification of Cystathionine β-Synthase (CBS), particularly truncated variants thereof. The present invention also relates to compositions of substantially pure CBS produced through said methods of purification.
Cystathionine β-synthase (CBS) plays an essential role in homocysteine (Hcy) metabolism in eukaryotes (Mudd et al., 2001, in The Metabolic and Molecular Bases of Inherited Disease, 8 Ed., pp. 2007-2056, McGraw-Hill, New York). The CBS enzyme catalyzes a pyridoxal 5′-phosphate (PLP; Vitamin B6)-dependent condensation of serine and homocysteine to form cystathionine, which is then used to produce cysteine by another PLP-dependent enzyme, cystathionine γ-lyase. In mammalian cells that possess the transsulfuration pathway, CBS occupies a key regulatory position between the remethylation of Hcy to methionine or its alternative use in the biosynthesis of cysteine. The relative flux between these two competing pathways is roughly equal and is controlled by intracellular S-adenosylmethionine (AdoMet) concentrations (Finkelstein and Martin, 1984, J. Biol. Chem. 259:9508-13). AdoMet activates the mammalian CBS enzyme by as much as 5-fold with an apparent dissociation constant of 15 μM (Finkelstein et al., 1975, Biochem. Biophys. Res. Commun. 66: 81-87; Roper et al., 1992, Arch. Biochem. Biophys. 298: 514-521; Kozich et al., 1992, Hum. Mutation 1: 113-123).
The C-terminal regulatory domain of human CBS consists of ˜140 amino acid residues (Kery et al., 1998, Arch. Biochem. Biophys. 355: 222-232). This region is required for tetramerization of the human enzyme and AdoMet activation (Kery et al., 1998, id.). The C-terminal regulatory region also encompasses the previously defined “CBS domains” (Bateman, 1997, Trends Biochem. Sci. 22: 12-13). These hydrophobic sequences (CBS 1 and CBS 2), spanning amino acid residues 416-468 and 486-543 of SEQ ID NO: 1, respectively, are conserved in a wide range of otherwise unrelated proteins. Their function remains unknown, although the sharp transition of thermally induced CBS activation and the observation that mutations in this domain can constitutively activate the enzyme indicates that they play a role in the autoinhibitory function of the C-terminal region (Janosik et al., 2001, Biochemistry 40: 10625-33; Shan et al., 2001, Hum. Mol. Genet. 10: 635-643; Miles and Kraus, 2004, J. Biol. Chem. 279: 29871-4). Two well-conserved CBS domains are also present in the C-terminal region of the yeast CBS, which is of approximately the same length as the human enzyme.
In healthy normal individuals, CBS-mediated conversion of Hcy to cystathionine is the rate-limiting intermediate step of methionine (Met) metabolism to cysteine (Cys). Vitamin B6 is an essential coenzyme for this process. In patients with certain genetic mutations in the CBS enzyme, the conversion of Hcy to cystathionine is slowed or absent, resulting in elevations in the serum concentrations of the enzymatic substrate (Hcy) and a corresponding decrease in the serum concentrations of the enzymatic product (cystathionine). The clinical condition of an elevated serum level of Hcy, and its concomitant excretion into the urine, is collectively known as homocystinuria.
Deficiency of CBS is the most common cause of inherited homocystinuria, a serious life-threatening disease that results in severely elevated homocysteine levels in plasma, tissues and urine. Estimates on the prevalence of homocystinuria vary widely. Ascertainment by newborn screening and clinical ascertainment have indicated a prevalence ranging from 1:200,000 to 1:335,000 (Mudd et al., 1995, The Metabolic and Molecular Basis of Inherited Diseases, McGraw-Hill: New York, p. 1279). The primary health problems associated with CBS-deficient homocystinuria (CBSDH) include: cardiovascular disease with a predisposition to thrombosis, resulting in a high rate of mortality in untreated and partially treated patients; connective tissue problems affecting the ocular system with progressive myopia and lens dislocation; connective tissue problems affecting the skeleton characterized by marfanoid habitus, osteoporosis, and scoliosis; and central nervous system problems, including mental retardation and seizures. Symptoms include dislocated optic lenses, skeletal disorders, mental retardation and premature arteriosclerosis and thrombosis (Mudd et al., 2001, id.). Homozygous CBS deficiency is associated with a multitude of clinical symptoms, including mental retardation, osteoporosis, kyphoscoliosis, stroke, myocardial infarction, ectopia lentis, and pulmonary embolism. Cardiovascular complications of the disease, in particular arterial and venous thrombosis, are the principal contributors to early mortality.
The pathophysiology of CBS deficiency is undoubtedly complex, but there is a consensus that the fundamental instigator of end-organ injury is an extreme elevation of serum Hcy, a substrate of CBS that builds-up in tissues and blood due to the absence of its CBS-catalyzed condensation with L-serine to form cystathionine. The toxicity of profound elevations in blood and tissue concentrations of Hcy may ensue from the molecular reactivity and biological effects of Hcy per se or from its metabolites (e.g. Hcy-thiolactone) that affect a number of biological processes (Jakubowski et al., 2008, FASEB J 22: 4071-6). Abnormalities in chronic platelet aggregation, changes in vascular parameters, and endothelial dysfunction have all been described in patients with homocystinuria.
Currently, three treatment options exist for the treatment of CB SDH:
Each of these three therapies is aimed at lowering serum Hcy concentration. The standard treatment for individuals affected with Vitamin B6 non-responsive CBSDH consists of a Met-restricted diet supplemented with a metabolic formula and Cys in the form of cysteine (which has become a conditionally essential amino acid in this condition). Intake of meat, dairy products, and other food high in natural protein is prohibited. Daily consumption of a poorly palatable, synthetic metabolic formula containing amino acids and micronutrients is required to prevent secondary malnutrition. Supplementation with betaine (trade name: Cystadane™, synonym: trimethylglycine) is also standard therapy, wherein betaine serves as a methyl donor for the remethylation of Hcy to Met catalyzed by betaine-homocysteine methyltransferase in the liver (Wilcken et al., 1983, N. Engl. J. Med. 309: 448-53). Dietary compliance generally has been poor, even in those medical centers where optimal care and resources are provided, and this non-compliance has major implications on the development of life-threatening complications of homocystinuria.
To enable patients with homocystinuria enjoy a far less restrictive diet (e.g. daily intake limited to 2 g protein per kg, which is easily attainable), and have a significantly decreased Hcy plasma level leading in the long-term to clinical improvement, a strategy for increasing enzyme activity provides potential for treatment as set forth in co-pending U.S. provisional patent application Ser. No. 61/758,138. The most effective therapeutic strategy is to increase enzyme activity, as is evident when Vitamin B6-responsive homocystinuria patients are given pyridoxone. However, this strategy is not possible for Vitamin B6 non-responsive patients due to the nature of the mutations. Enzyme replacement therapy (ERT) as a way to increase enzyme activity in these patients requires exogenous enzyme, which is not present in the art and thus raises a need in the art for improved reagents and methods for producing CBS in greater yields of sufficiently purified enzyme for therapeutic administration.
Kraus and colleagues have developed expression systems and fermentation conditions for generating active recombinant human CBS and variants thereof (U.S. Pat. Nos. 5,635,375, 5,523,225 and 7,485,307, incorporated by reference herein in their entireties for any purpose). These proteins were purified by processes relevant for academic purposes, including use of protein leads on the proteins which are not considered useful for preparation of pharmaceuticals.
In order to employ methods of increasing CBS enzyme activity, an efficient method of CBS enzyme purification is required. Existing methods of purification for recombinant CBS protein rely on affinity tags to facilitate purification that does not provide the desired purity and efficiency. Therefore to more efficiently obtain the necessary levels of CBS required for therapeutic use there is a need for improved downstream purification of CBS protein produced in microbial cells.
This invention provides methods for purifying cystathionine β-Synthase (CBS), wherein said CBS protein is a naturally occurring truncated variant, or a chemically cleaved or genetically engineered truncate thereof, and particularly truncated CBS produced in recombinant cells. In 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 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.
In certain embodiments the method further comprises performance of additional chromatographic steps (known in the art as “polishing” steps). In particular embodiments, the methods of the invention include the step of performing chromatographic separation using a Hydrophobic Interaction Chromatography (HIC) column. In other embodiments the method further comprises the step of performing chromatographic separation using a ceramic hydroxyapaptite resin.
In certain embodiments the ion exchange column is an anion exchanger, preferably a weak anion exchanger. In particular embodiments the anion exchanger is a DEAE-Sepharose FF column. In further embodiments the IMAC resin is charged with a divalent ion. In yet further embodiments the divalent metal ion is nickel, copper, cobalt or zinc. In more specific embodiments the divalent metal ion is zinc.
In certain other embodiments the method further comprises eluting CBS from the IMAC resin with an elution buffer comprising imidazole. In certain embodiments the CBS-containing solution is a clarified CBS solution, wherein cell debris and other particulate matter is removed from a suspension comprising CBS including but not limited to supernatant after centrifugation or filtrate after filtration. In yet other embodiments the CBS-containing solution is obtained by homogenizing cells expressing a recombinant construct comprising a nucleic acid sequence encoding CBS. In certain embodiments the CBS nucleic acid sequence comprises SEQ ID NO. 1 and encodes a protein have the amino acid sequence identified as SEQ ID NO: 2. In other embodiments the nucleic acid sequence is truncated. In yet other embodiments the truncated CBS nucleic acid sequence 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 In other certain embodiments the recombinant cells are microbial cells, particularly bacterial cells. In particular embodiments, the bacterial cells are E. coli cells, particularly recombinant E. coli cells that produce a mammalian, preferably human, CBS protein. In certain particular embodiments, said human CBS protein has an amino acid sequence as set forth in SEQ ID NO:3 or a truncated CBS nucleic acid sequence that has been truncated to an ending position of one of amino acid residues from 382-532 or 543-550 of SEQ ID NO:2. In other particular embodiments, the truncated CBS nucleic acid sequence is optimized for expression in E. coli, identified by SEQ ID NO: 4.
In another aspect, a substantially purified CBS solution is provided using a method comprising the steps of: a) providing a CBS-containing solution in the presence of at least one impurity, wherein said CBS protein is a naturally occurring truncated variant, or a chemically cleaved or genetically engineered truncate thereof, and particularly truncated; and (b) performing chromatographic separation of said CBS-containing solution using a metal affinity chromatography (IMAC) resin. In additional particular embodiments, a substantially purified CBS solution is provided using a method comprising 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.
In certain embodiments of the invention the substantially purified CBS solution is formulated in a pharmaceutically acceptable carrier.
In another aspect, the invention provides methods for producing an enriched CBS solution, the method comprising of: (a) providing a CBS-containing solution in the presence of at least one impurity, wherein said CBS protein is a naturally occurring truncated variant, or a chemically cleaved or genetically engineered truncate thereof, and particularly truncated; and (b) performing chromatographic separation of said CBS-containing solution using an immobilized metal affinity chromatography (IMAC) resin charged with a divalent metal ion.
In another aspect, an enriched CBS solution is provided using a method comprising the steps of: a) providing a CBS-containing solution in the presence of at least one impurity, wherein said CBS protein is a naturally occurring truncated variant, or a chemically cleaved or genetically engineered truncate thereof, and particularly truncated; and (b) performing chromatographic separation of said CBS-containing solution using an immobilized metal affinity chromatography (IMAC) resin charged with a divalent metal ion.
It is a particular advantage of this invention that purification of recombinant, full-length or truncated CBS, particularly human CBS, can be achieved without further modification of the protein, e.g., by incorporating a “tag” molecule known in the art (poly-HIS, FLAG, etc.). Use of the chromatographic methods disclosed herein advantageously makes these tags unnecessary, thus avoiding additional recombinant manipulation and any disadvantages (in immunogenicity, in vivo half-life or biochemical activity) that might be introduced into any preparation of recombinant CBS containing such a tag.
Specific preferred embodiments of the invention will become evident from the following more detailed description of certain preferred embodiments and the claims.
The following detailed description of the embodiments of the present invention can be best understood when read in conjunction with the following drawings.
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.
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.
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.
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.
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 (Ca10(PO4)6(OH)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.
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).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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).
As used herein an enriched CBS solution is a solution subjected to one or more purification steps.
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:
Specific Activity Final Fraction/Specific Activity Crude Fraction.
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:
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.
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.
Each reference described and/or cited herein is incorporated by reference in its entirety.
The following examples are provided for the purpose of illustration and are not intended to limit the scope of the present invention.
A truncated human CBS variant lacking specific portions of the non-conserved regions (r-hCβSΔC; 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-hCβSΔC (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.
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.
Bacterial Expression Analysis of CBS Deletion Mutants-Growth of E. coli.
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 μg/mL ampicilin and 0.001% thiamine in the presence or absence of 0.3 mM δ-aminolevulinate (δ-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.
CBS Activity Assay—
CBS activity was determined by a previously described radioisotope assay using [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 μmol of cystathionine in 1 h at 37° C.
Denaturing and Native Polyacrylamide Gel Electrophoresis and Western Blotting—
Western 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% bromphenol 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).
Densitometric Scanning Analysis—
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.
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-hCβSΔC; 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 (48,000×g) 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.
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 H2O to approach the ionic strength of the equilibration/wash buffer (50 mM NaCl), and elution with 137 mM NaCl (
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.
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. (
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 A280 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. (
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 (NH4)2SO4. 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 (NH4)2SO4. The non-binding flow through of this column experiment was collected and adjusted to 1.25M (NH4)2SO4, and reloaded on to a column equilibrated to the same concentration of (NH4)2SO4. In this case there was evidence of significant binding to the column. A 20 column volume (NH4)2SO4 gradient elution was performed from 1.25M to 0.25M (NH4)2SO4 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 (NH4)2SO4 determined the final operational parameters. (
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 A280) 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. (
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 (
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.
This application is a non-provisional application of U.S. Provisional Application No. 61/615,629, filed Mar. 26, 2012, the disclosure of each of which is incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61615629 | Mar 2012 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13830494 | Mar 2013 | US |
| Child | 14970814 | US |
