Efficient and rapid purification of recombinant human α-galactosidase A by affinity column chromatography

Efficient and rapid purification of recombinant human α-galactosidase A by affinity column chromatography

Protein Expression and Purification 37 (2004) 499–506 www.elsevier.com/locate/yprep Efficient and rapid purification of recombinant human a-galactosidase...

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Protein Expression and Purification 37 (2004) 499–506 www.elsevier.com/locate/yprep

Efficient and rapid purification of recombinant human a-galactosidase A by affinity column chromatography Kayo Yasuda, Hui-Hwa Chang, Hui-Li Wu, Satoshi Ishii1, Jian-Qiang Fan* Department of Human Genetics, Mount Sinai School of Medicine, Box 1498, Fifth Avenue at 100th Street, New York, NY 10029, USA Received 21 June 2004, and in revised form 4 July 2004 Available online 12 August 2004

Abstract The lysosomal enzyme a-galactosidase A (a-Gal A) metabolizes neutral glycosphingolipids that possess a-galactoside residues at the non-reducing terminus, and inherited defects in the activity of a-Gal A lead to Fabry disease. We describe here an efficient and rapid purification procedure for recombinant a-Gal A by sequential Concanavalin A (Con A)–Sepharose and immobilized thio-agalactoside (thio-Gal) agarose column chromatography. Optimal elution conditions for both columns were obtained using overexpressed human a-Gal A. We recommend the use of a mixture of 0.9 M methyl a-mannoside and 0.9 M methyl a-glucoside in 0.1 M acetate buffer (pH 6.0) with 0.1 M NaCl for the maximum recovery of glycoproteins with multiple high-mannose type sugar chains from Con A column chromatography, and that the Con A column should not be reused for the purification of glycoproteins that are used for structural studies. Binding of the enzyme to the thio-Gal column requires acidic condition at pH 4.8. A galactose-containing buffer (25 mM citrate–phosphate buffer, pH 5.5, with 0.1 M galactose, and 0.1 M NaCl) was used to elute a-Gal A. This procedure is especially useful for the purification of mutant forms of a-Gal A, which are not stable under conventional purification techniques. A protocol that purifies an intracellular mutant a-Gal A (M279I) expressed in COS-7 cells within 6 h at 62% overall yield is presented.  2004 Elsevier Inc. All rights reserved. Keywords: a-Galactosidase A; Concanavalin A; Affinity column chromatography; Fabry disease; Active-site specific chaperone; Enzyme purification

a-Galactosidase (EC 3.2.1.22) is widely distributed in nature and is found in bacteria, plants, and mammals [1–3]. In humans, lysosomal a-galactosidase A (a-Gal A) is responsible for the metabolism of neutral glycosphingolipids that possess a-galactosyl residues at the non-reducing terminus, predominantly globotriaosylceramide (Gb-3). Deficiency in enzyme activity results in Fabry disease, a lysosomal storage disorder caused by the accumulation of glycosphingolipids in body fluids and tissue lysosomes [4]. Patients typically succumb to Fabry disease between the fourth and fifth decades of *

Corresponding author. Fax: 1 732 745 9769. E-mail address: [email protected] (J.-Q. Fan). 1 Present address: Department of Agricultural and Life Sciences, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan. 1046-5928/$ - see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2004.07.005

life because of vascular disease of the heart, brain, and/or kidneys. Enzyme replacement therapy, which involves the infusion of purified human wild-type enzyme into patients, has recently become available. To date, over 270 a-Gal A mutations that result in Fabry disease have been described (The Human Gene Mutation Database, Cardiff). These mutations are highly heterogeneous and most of them are unique to a single affected family. More than 70% of all disease-causing mutations are single nucleotide substitutions that often result in missense mutations in a-Gal A, and many of the mutant enzymes retain residual enzyme activity in patient cells [4]. We have proposed a small moleculebased active-site specific chaperone (ASSC) therapy for Fabry disease [5]. The approach is effective in patient cells and an animal model, particularly for those missense mutations that result in misfolding of the mutant

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protein and subsequent excessive degradation of the mutant protein in the endoplasmic reticulum associated degradation (ERAD) [6]. Recently, the X-ray crystal structure of human a-Gal A was revealed and the relationship between molecular defect of various mutations and disease phenotypes was discussed [7]. However, only limited information on the enzymatic and physiological properties of mutant enzymes can be obtained, because of the difficulty in efficiently expressing and purifying mutant enzymes. Successful expression and purification of wild-type aGal A from different sources, including human tissues [8], stably expressed Chinese hamster ovary (CHO) cells [9], culture medium of baculovirus infected insect cells [10,11], and yeast [12,13], has been demonstrated. Enzymes purified from the culture medium of recombinant CHO cells and gene-activated human fibroblasts are used as therapeutic agents (Fabrazyme and Replagal, respectively) for the treatment of Fabry disease [14]. Purification typically involves multiple steps of column chromatography, including ion exchange and/or hydrophobic chromatography. Since mutant enzymes are frequently less stable than the wild-type enzyme [15,16], the purification protocol developed for the wild-type a-Gal A may not be sufficient for mutant a-Gal A enzymes. Therefore, a rapid and efficient procedure for the purification of mutant forms of a-Gal A needs to be developed. In this report, we present an efficient expression and rapid purification procedure for both recombinant wild-type and mutant a-Gal A using affinity column chromatography. A detailed purification protocol is also presented, demonstrating that an intracellular a-Gal A mutant (M296I) overexpressed in COS-7 cells can be efficiently purified within 6 h at 62% yield. This purification protocol is readily scalable for the purification of large volumes of recombinant wild-type a-Gal A that can be used in enzyme replacement therapy for Fabry disease.

Materials and methods Site-directed mutagenesis of a-Gal A Expression vectors containing Fabry mutations were generated by site-directed PCR-mutagenesis [17] using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Two amino acid substitutions, R301Q (Arg-301 fi Gln; CGA fi CAA) and M296I (Met-296 fi Ile; ATG fi ATA), were introduced individually by PCR amplification performed with PfuTurbo DNA polymerase, wild-type a-Gal A sequence as a template, and a primer set of 5 0 -CATGTCTAATGACC TCCAACACATCAGCCCTCAAG-3 0 and 5 0 -CTTGA GGGCTGATGTGTTGGAGGTCATTAGACATG-3 0 for R301Q mutation, and 5 0 -GGCTGCTCCTTTATTC ATATCTAATGACCTCCGAC-3 0 and 5 0 -GTCGGAG

GTCATTAGATATGAATAAAGGAGCAGCC-3 0 for M296I mutation, respectively. Following digestion with DpnI, the nicked DNA vector carrying the desired mutation was transformed into E. coli, and clones expressing the mutant sequence were selected. Each vector was sequenced to confirm the desired mutation before it was further subcloned into the expression vector pCXN2 [15]. Final constructs were designated pCXN2-GLA-R301Q and pCXN2-GLA-M296I, respectively. These expression plasmids were used to generate mammalian mutant enzymes in transfected COS-7 cells. Overexpression of human a-Gal A in Sf9 cells infected with recombinant baculovirus Recombinant baculoviruses that express either wildtype or R301Q mutant a-Gal A were generated by the Bac-To-Bac Baculovirus Expression System [18] (Life Technologies, Gaithersburg, MD). Plasmid DNA sequences coding either wild-type or R301Q mutant a-Gal A were cloned into a pFastBac donor plasmid, and the recombinant plasmids were transformed into DH10Bac competent cells which contain a baculovirus shuttle vector (Bacmid) driven by a bacurovirus-specific promoter (AcNPV), and then further transposed to form a recombinant baculovirus shuttle vector (recombinant Bacmid). A stock of recombinant baculovirus was obtained by transfection of Spodoptera frugiperda (Sf9) cells with the recombinant Bacmid, followed by purification using series dilution method on 96-well plates. Further infection of suspended Sf9 cells, cultured in Sf-900II medium (Life Technology) supplemented with 1% penicillin–streptomycin at 28 C, with the baculovirus stock solution provided overexpressed a-Gal A in the culture medium. Transient expression of a-Gal A in COS-7 cells COS-7 cells (0.8 · 106) were grown in 10-cm tissue culture dishes in DulbeccoÕs Modified EagleÕs medium supplemented with 10% fetal calf serum and 1% penicillin–streptomycin at 37 C, 5% CO2. Three microgram of plasmid DNA was transfected into COS-7 cells using 9 ll FuGene6 (Roche, Indianapolis, IN), and the cells were cultured in medium containing 1-deoxy-galactonojirimycin (DGJ, Toronto Research Chemicals, Toronto, Canada) at 20 lM as an ASSC for three days. The cells were rinsed with phosphate-buffered saline and homogenized in water with a homogenizer (Niti-on, Chiba, Japan). The supernatant obtained after centrifugation at 10,000g was used as an enzyme source. Enzyme assay and protein determination a-Gal A activity was assayed with 4-methylumbelliferyl a-D -galactoside (4MU-a-Gal, Sigma Chemical, St. Louis, MO) or p-nitrophenyl-a-galactoside (pNP-a-Gal,

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Sigma) as substrate. Comparing to using pNP-a-Gal, the assay using 4MU-a-Gal is much more sensitive and suitable for the determination of intracellular enzyme activity. A typical 4MU-a-Gal assay was performed with a reaction mixture (60 ll) containing 0.1 M sodium citrate buffer (pH 4.6), 5 mM 4MU-a-Gal, 75 mM N-acetylgalactosamine as an inhibitor for a-galactosidase B (a-N-acetylgalactosaminidase) and enzyme source at 37 C for 10 min, followed by termination of the enzyme reaction by addition of 1.2 ml of 0.1 M glycine–NaOH buffer (pH 10.7). The released 4-methylumbelliferone was determined by fluorescence measurement at 360 and 450 nm as excitation and emission wavelengths, respectively. One unit of enzyme activity was defined as the amount of enzyme that releases 1 nmol of 4-methylumbelliferone per hour. When pNP-a-Gal was used as substrate, a-Gal A activity was determined in 150 ll reaction mixture composed by 2 mM pNP-a-Gal and the enzyme in 25 mM citrate buffer (pH 4.6). Following the addition of 700 ml of 0.2 M borate buffer (pH 9.8), the released p-nitrophenol was quantitatively determined by absorbance at 400 nm. Since pNP-a-Gal is typically used for assaying a-Gal A in an overexpression system (e.g., insect cell/baculovirus system), the inhibitor (N-acetylgalactosamine) for a-galactosidase B was not included. One unit of enzyme activity using pNP-a-Gal was defined as the amount of enzyme that releases 1 lmol of p-nitrophenol per min. The protein concentration was determined using a DC Protein Assay kit with bovine serum albumin (BSA) as a standard (both from Bio-Rad Laboratories, Hercules, CA). SDS–polyacrylamide gel electrophoresis SDS–polyacrylamide gel electrophoresis (SDS– PAGE) was performed by the method of Laemmli [19], and proteins were visualized by a silver stain kit (Bio-Rad). Molecular weight markers composed of phosphorylase (97.4 kDa), serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), and trypsin inhibitor (21.5 kDa) were obtained from BioRad Laboratories. A purification protocol for mutant a-Gal A by affinity columns Mutant a-Gal A was transiently overexpressed in COS-7 cells, and the cell lysate was used as an enzyme source for purification. Con A–Sepharose and immobilized thio-a-galactose (thio-Gal) agarose were purchased from Sigma and Pierce Biotechnology (Rockford, IL), respectively. All purification steps were performed at 4 C. Con A–Sepharose chromatography An equal volume of Washing Buffer (0.1 M sodium acetate buffer, pH 6.0, containing 0.1 M NaCl, 1 mM

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MgCl2, 1 mM CaCl2, and 1 mM MnCl2) was added to the cell lysate (2 ml) from 10 10-cm culture dishes, and then applied to a Con A–Sepharose column (0.5 · 7 cm) equilibrated with Washing Buffer. After washing with Washing Buffer (approximately 15 volumes of the column), the enzyme was eluted with 30 ml Elution Buffer I (0.9 M Me-a-Man and 0.9 M Me-a-Glc in Washing Buffer). Typically, the enzyme was eluted out within less than 15 ml of the Elution Buffer I. Buffer exchange by ultrafiltration An ultrafiltration device (Centricon Plus-20 with 10 kDa molecular cutoff, Millipore, Bedford, MA) was used for the removal of highly concentrated methyl monosaccharides, and for the exchange of buffer to an acidic one to allow efficient binding of the mutant enzyme to a thio-Gal agarose column. To avoid unnecessary non-specific binding of the protein, bovine serum albumin (0.5 mg in 5 ml H2O) was added to the Centricon tube and briefly centrifuged prior to loading of the sample. The fractions containing high a-Gal A activity after separation on the Con-A column were combined and added to the Centricon tube, and the total volume was topped to 19 ml with 25 mM citrate–phosphate buffer (pH 4.8) containing 0.1 M NaCl. Centrifugation at 2400g for 30 min at 4 C typically reduced the volume to 3 ml. The Centricon tube was then filled with the citrate–phosphate buffer up to 19 ml and centrifuged at 2400g for 20 min at 4 C. This procedure was repeated one more time, and the final preparation was collected in 3 ml, which was then applied to a thio-Gal affinity column as described below. Thio-Gal affinity column chromatography The sample was applied to a thio-Gal agarose column (0.9 · 5.5 cm) equilibrated with Binding Buffer (25 mM citrate–phosphate buffer, pH 4.8, containing 0.1 M NaCl). After washing the column with six volumes of the same buffer, the enzyme was recovered by eluting with 20 ml Elution Buffer II (25 mM citrate–phosphate buffer, pH 5.5, containing 0.1 M NaCl and 0.1 M galactose) and concentrated by ultrafiltration with the Centricon device.

Results and discussion Overexpression of mutant a-Gal A The mutations R301Q and M296I are found in cardiac variant Fabry patients with 4 and 9% residual enzyme activity, respectively [20,21]. To characterize the mutant a-Gal A enzymes with these missense mutations, recombinant plasmids were constructed to produce human mutant a-Gal A in transfected COS-7 cells. Mutant

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a-Gal A enzymes with several different missense mutations have been expressed in transfected COS-7 cells at a lower level compared with the expression of wild-type enzyme [15,16]. We hypothesized that the cardiac mutations cause a deficiency in enzyme activity primarily because of misfolding of the mutant enzyme in the early biosynthetic pathway and premature degradation of the protein in the ERAD, and have shown that inclusion of DGJ into the culture medium prevents degradation and accelerates the maturation of the mutant protein [6,22]. DGJ is a potent inhibitor of a-Gal A, however, at subinhibitory concentrations, it is also an effective ASSC which corrects misfolding or induces proper folding of mutant a-Gal A in the ER to prevent excessive degradation of the mutant protein in the ERAD [5]. When the plasmid DNAs were transfected into COS7 cells, the enzyme activities in the transfected cells were found to be 699 and 3768 U/mg protein with pCXN2GLA-R301Q and pCNX2-GLA-M296I, respectively (Table 1). The higher enzyme activity for the M296I mutation could be attributed to the nature of the mutation, since residual enzyme activity in M296I lymphoblasts is higher than in R301Q lymphoblasts. To maximize the production of these mutant enzymes, DGJ was added to the culture medium, resulting in a 3.1- and 1.8-fold increase in enzyme activity for the R301Q and M296I mutations, respectively (Table 1). The enzyme activity was further shown to be 4728 and 11,894 U/mg protein for these two mutations, respectively, upon removal of residual DGJ by ultrafiltration of the cell lysate using Centricon (molecular cutoff at 10 kDa). This result suggests that both mutant enzymes suffer from excessive degradation in COS-7 cells, and that inclusion of DGJ can significantly increase production of mutant enzymes. Because enzyme activity in transfected COS-7 cells cultivated with DGJ was approximately 20–49-fold higher than endogenous COS-7 a-Gal A activity, the enzyme prepared from transfected COS-7 cells can be readily used for the characterization of human mutant a-Gal A. Although the COS-7 expression system is not

Table 1 Expression of human mutant a-Gal A in transfected COS-7 cells Mutation

COS-7 Wild-type R301Q M296I

Enzyme activity (U/mg) DGJ

+DGJ

196 ± 10.9 6707 ± 717 699 ± 88 3768 ± 409

245 ± 1.4 6987 ± 484 2142 ± 252 6964 ± 249

Increase (fold)

1.3 1.0 3.1 1.8

COS-7 cells were transfected with either pCXN2-GLA-R301Q or pCXN2-GLA-M296I. The cells were cultured in the presence or absence of DGJ (20 lM in culture medium) for three additional days after transfection. Cells were lysed in water, and a-Gal A activity in cell homogenates was assayed using 4MU-a-Gal as substrate. The results were an average of three samples.

suitable for large scale production, it is convenient for producing various mutant enzymes on a small scale. Insect cells have been used for the efficient production of wild-type a-Gal A using recombinant baculovirus [10,11]. The R301Q mutant a-Gal A was effectively secreted into the culture medium of insect cells at approximately 7.5 mg/L. This may be due to the lower culture temperature (28 C) for insect cells, promoting a more stable conformation of the mutant enzyme, thereby preventing degradation of the enzyme in the ERAD. The enzyme produced in insect cells may not be suitable for therapeutic use because of different glycosylation sets between insect cells and mammalian cells [24]. The insect overexpression system, though, can be useful for the production of large quantities of mutant enzymes when the conformational properties of these enzymes need to be characterized. Evaluation of elution conditions of glycoprotein for Con A–Sepharose column Con A is a carbohydrate-binding, legume-derived lectin that recognizes glycoproteins and glycopeptides containing high-mannose type glycans, as well as hybrid-type and bi-antennary glycans [24]. Crystallographic studies indicate that the lectin preferably binds the Mana1 fi 2Man structure in sugar chains [25] and recognizes in particular high-mannose type sugar chains in glycoproteins [26]. In most previous reports, 0.5 M Me-a-Man was used for eluting bound proteins from the affinity column. Recently, a quantitative recovery of Man9GlcNAc2Asn derivatives from a Con A–Sepharose column was achieved by eluting with 1 M Me-aMan [27]. However, we have observed that these elution methods are not sufficient for the full recovery of glycoprotein (data not shown). Since the Con A–Sepharose column is the most widely used affinity column for purification of glycoproteins, it is important to optimize elution conditions. Human a-Gal A expressed from an insect cell/baculovirus expression system was used to examine the elution condition, because of the fact that major N-glycans on insect cell-derived glycoproteins contain terminal mannose residues [23] which could result in tight binding to the column. When eluting with 0.5 M Me-a-Man, only 41% of a-Gal A was recovered from the column (Fig. 1). Additional a-Gal A, accounting for 38% of total enzyme activity, was eluted with 1.0 M Me-a-Man using the procedure recommended previously [27]. Unexpectedly, more a-Gal A, accounting for 10% of total enzyme activity, was eluted with a buffer containing 0.9 M Me-a-Man and 0.9 M Me-a-Glc. The amount of the methyl monosaccharides is near the saturation concentration of the sugars at 4 C. Mature a-Gal A contains four potential N-glycosylation sites and three are glycosylated in protein overex-

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Fig. 1. Evaluation of Con A–Sepharose column chromatography elution conditions. Human a-Gal A was expressed in Sf-9 cells infected with recombinant baculovirus. Following the removal of cells by centrifugations, the supernatant (approximate 2 L) was applied to a Con A–Sepharose column (1.5 · 7 cm) equilibrated with Washing Buffer at 4 C. After washing the column with the same buffer until the 280 nm absorbance fell below 0.01, a-Gal A was sequentially eluted with: (a) 0.5 M Me-a-Man in Washing Buffer; (b) 1.0 M Me-a-Man in Washing Buffer; and (c) 0.9 M Me-a-Man and 0.9 M Me-a-Glc in Washing Buffer. Each 5.5 ml of eluent was collected in an individual tube. a-Gal A activity was determined in 25 mM citrate buffer (pH 4.6) using pNP-a-Gal as a substrate. Bars on the top of the chromatogram indicate the tubes that were combined for the enzyme fractions.

pressed from CHO cells [28]. The multiple high-mannose type N-glycosides in a-Gal A may contribute to the tighter binding of a-Gal A to the Con A–Sepharose column, compared to the binding of soybean agglutinin peptide which contains a single carbohydrate chain per subunit [27]. Although we recovered more than 90% of a-Gal A that was loaded onto the column by eluting with a buffer containing 0.9 M Me-a-Man and 0.9 M Me-a-Glc, this elution condition may not be suitable for other highly glycosylated proteins, particularly for those glycoproteins with multiple high-mannose type glycans, because the affinity of these proteins may be greater than that of a-Gal A expressed in baculovirusinfected insect cells. For this reason, we recommend that Con A–Sepharose not be reused for the purification of highly glycosylated proteins, especially when used for the structural determination of glycosylation or examination of the biological function of proteins. Optimization of chromatography for the purification of mutant a-Gal A Several mutant forms of a-Gal A have been reported to be less stable than the wild-type enzyme [15,16]. These mutants may lose significant enzyme activity during conventional purification. The R301Q mutant a-Gal A enzyme known to be unstable [6] was expressed in an insect cell/baculovirus expression system and used to de-

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velop an efficient purification strategy. Since a-Gal A is a glycoprotein, a Con A–Sepharose column was used in the first purification step. The culture medium, containing approximately 75 mg of protein, was loaded onto a Con A–Sepharose column equilibrated with Washing Buffer. Following a wash with Washing Buffer, the enzyme was eluted with Elution Buffer I (0.9 M Me-aMan and 0.9 M Me-a-Glc in Washing Buffer). The mutant a-Gal A efficiently bound to the Con A–Sepharose column, and was eluted with Eluting Buffer I with a recovery of 64% (Fig. 2A). The lower yield may be attributed to the instability of the mutant enzyme. This result indicates that the Con A–Sepharose column is effective and efficient as the first step in the separation of mutant a-Gal A from culture medium. To avoid unnecessary loss of mutant a-Gal A activity, we chose enzyme-specific affinity column chromatography for further purification. Affinity purification of a-galactosidase has been performed using derivatives of the substrate analog, such as a-D -galactosylamine derivatives [29,30]. Recently, hybrid affinity chromatography using a substrate analog and immobilized metal affinity matrix as ligand was developed for the purification of a-galactosidase from the roots of Verbascum thapsus [31]. Since these methods involve extensive in-house chemical syntheses of ligands, we examined a variety of commercially available affinity resins. Among them, an immobilized D -galactose cross-linked agarose gel (Pierce) gave the most satisfactory result. The galactosyl residue is a-configured and linked via a thio-ether to an aglycon that is in turn linked to the gel matrix (communication with Pierce technical support). A paminophenyl a-D -Gal agarose gel (Sigma) was also able to bind the enzyme. However, the capacity of the p-aminophenyl a-D -Gal resin was about 30-fold lower than that of the thio-Gal gel (data not shown). The mutant a-Gal A (R301Q) eluted from the Con A column was used to determine the proper elution conditions from the thio-Gal column. Since lysosomal a-Gal A has a narrow active pH at 4.0–5.0, and a stable pH at 4.5–6.0 [4], most of the enzyme is expected to be bound to the column at pH 4.5–5.0. Partially purified R301Q a-Gal A (70,000 U) was loaded onto a thioGal column (0.5 · 7.5 cm) with Binding Buffer (25 mM citrate–phosphate buffer, pH 4.8, and 0.1 M NaCl). A small amount of the enzyme leaked out of the column (Fig. 2B), probably because of column over-loading, since the flow-through protein was able to bind to a second thio-Gal column. In fact, a smaller quantity of enzyme (45,000 U) completely bound to the column, indicating that the capacity of the thio-Gal column is at least 30,000 U/ml (15 lg a-Gal A/ml) under this condition. The enzyme was eluted from the thio-Gal column with Elution Buffer II (Fig. 2B). Over 80% of enzyme activity was recovered from the column. Elution with

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Example of purification of mutant a-Gal A expressed in mammalian cells The purification of intracellular proteins from mammalian cells is often more challenging than those secreted into culture medium. In addition to the purification of a-Gal A from the culture medium of insect cells

Fig. 2. Optimization of column chromatography for mutant a-Gal A purification. Culture medium (20 ml) of Sf-9 cells infected with recombinant baculovirus encoding R301Q a-Gal A cDNA was used as starting material. a-Gal A activity was determined in 0.1 M citrate buffer (pH 4.6) using 4MU-a-Gal as substrate. All column preparations were done at 4 C. (A) Con A–Sepharose column chromatography. Sample was loaded to a Con A–Sepharose column (0.5 · 7 cm) pre-equilibrated with Washing Buffer. Mutant a-Gal A was eluted with 0.9 M Me-a-Man and 0.9 M Me-a-Glc in Washing Buffer. Each 4.8 ml of eluent was collected in an individual tube. (B) Thio-Gal affinity column chromatography. Partially purified R301Q a-Gal A (70,000 U) was applied to a thio-Gal agarose column (0.5 · 7.5 cm) equilibrated with Binding Buffer at 4 C. The enzyme was eluted with 0.1 M galactose in Binding Buffer at pH 5.5. Each 1.2 ml of eluent was collected in an individual tube. Arrows and bars on the top of the chromatogram indicate the position of a buffer change, and the tubes were combined to give an enzyme fraction, respectively. (e) absorbance at 280 nm; ( ) enzyme activity.



Elution Buffer II at pH 5.5 without inclusion of galactose resulted in a broad elution pattern, suggesting that galactose is efficient in eluting a-Gal A. The enzyme purified by thio-Gal column chromatography was shown to be a single 46 kDa band by SDS–PAGE and silver staining (data not shown), indicating that Con A and thio-Gal column chromatographies are sufficient for purification of a-Gal A expressed in an insect cell/ baculovirus expression system.

Fig. 3. Purification of mutant a-Gal A expressed in transfected COS-7 cells by affinity column chromatography. Human mutant a-Gal A (M296I) was expressed in 10 10-cm culture dishes of COS-7 cells transfected with pCXN2-GLA-M296I. The cell lysates were combined as the enzyme source. Enzyme activity was determined in 0.1 M citrate buffer (pH 4.6) using 4MU-a-Gal as substrate. (A) Con-A chromatography. The cell lysate was loaded onto a Con A–Sepharose column (0.5 · 7 cm) equilibrated with Washing Buffer, and eluted with 0.9 M Me-a-Man and 0.9 M Me-a-Glc in Washing Buffer. Each fraction was collected as 2.5 ml eluent. (B) Thio-Gal affinity chromatography. Sample was loaded onto a thio-Gal column (0.9 · 5.5 cm) equilibrated with 25 mM citrate–phosphate buffer (pH 4.8) containing 0.1 M NaCl. The enzyme was eluted with 0.1 M galactose in the same buffer (pH 5.5). One fraction contained 2.2 ml eluent. Arrows and bars on the top of the chromatogram indicate the positions of a buffer change, and the tubes were combined to give an enzyme fraction, respectively. (e) Absorbance at 280 nm; ( ) enzyme activity.



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Fig. 4. Homogeneity of purified mutant a-Gal A. Samples were taken from preparations of M296I a-Gal A expressed in transfected COS-7 cells. SDS–PAGE was performed and gels were visualized with a silver stain kit. Lane 1, cell lysate; lane 2, enzyme preparation after Con A purification; and lane 3, enzyme preparation after thio-Gal affinity chromatography.

infected with recombinant baculovirus, as described above, we developed a detailed protocol for the efficient purification of intracellular a-Gal A from COS-7 cells transfected with mutant a-Gal A cDNA, as described in the Materials and methods Section. Mutant a-Gal A cDNA with the M296I mutation was transfected into COS-7 cells, and the enzyme was expressed in cells cultured in 10-cm culture dishes. The M296I mutation is clinically important because it causes the cardiac variant phenotype of Fabry disease, patients with which typically have about 9% of normal enzyme activity [23]. DGJ was added to the culture medium as an ASSC to increase the production of mutant enzyme. After twice washing the cells with phosphate-buffered saline, the cell lysate was combined and used as a source of enzyme. The cell lysate was applied to a Con A–Sepharose column equilibrated with Washing Buffer, and the enzyme was eluted with Elution Buffer I (Fig. 3A). This step is essential to remove most of the intracellular pro-

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teins, as revealed by SDS–PAGE (Fig. 4). Because high concentrations of methyl monosaccharides interfere with the interaction between the enzyme and second affinity column (the thio-Gal column), ultrafiltration using Centricon tubes was performed three times to sufficiently reduce the amount of methyl monosaccharides. Changing the buffer to an acidic one, which is required for enzyme binding, was also effectively accomplished by this procedure. To avoid non-specific absorption of protein by the membrane in the device, we strongly recommend blocking the membrane with bovine albumin or any other protein before loading the sample. Otherwise, this could result in a substantial impact on the overall yield, especially for samples with low protein concentration. After the ultrafiltration, the mutant enzyme solution (3 ml) was applied to a thio-Gal agarose column equilibrated with 25 mM citrate–phosphate buffer (pH 4.8) containing 0.1 M NaCl. The enzyme was recovered by eluting with 25 mM citrate–phosphate buffer (pH 5.5) containing 0.1 M NaCl and 0.1 M galactose (Fig. 3B), followed by concentration using ultrafiltration. The final sample was seen as a single band on a silver-stained SDS–PAGE gel (Fig. 4). No protein could be detected by the DC Protein Assay Kit (Bio-Rad, CA) in the combined fractions after Con A–Sepharose and thio-Gal chromatographies, indicating the efficiency of these affinity columns. The final specific enzyme activity was estimated to be 2000 U/lg protein, based on the protein concentration determined from a dilution series of BSA on a silver stained SDS–polyacrylamide gel. This purification protocol can be accomplished within 6 h with an overall yield of 62% (Table 2). In conclusion, we have described an efficient and rapid purification method for a-Gal A, particularly useful for mutant forms of the enzyme as exemplified with two different mutants. We have used this purification procedure to obtain various a-Gal A mutants in sufficient quantity to study their kinetic and physiological properties (the results will be published elsewhere). Although the protocol described here is for the preparation of small amounts of protein, the purification procedure could be scalable for large amount purification. Enzyme replacement therapy has proven to be effective for Fabry

Table 2 Summary of purification of intracellular mutant a-Gal A in transfected COS-7 cells Step

Total protein (mg)

Total enzyme activity (U)

Specific activity (U/mg)

Recoveryb (%)

Purification fold

Cell lysate Con A–Sepharose column chromatography Thio-Gal column chromatography

14.3 n.d.

43888 33832

3,069 —

100 77

1 —

0.012a

25556

2,129,667

62

694

All purification steps were performed at 4 C. Enzyme activity was determined after combining fractions with enzyme activity determined using 4MUa-Gal as a substrate. Protein was determined using the DC Protein Assay kit unless specified.n.d., not detectable. a Amount estimated by band density on SDS–PAGE with a silver staining method using BSA as reference protein. b Recovery was estimated from total enzyme activity.

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disease, but this therapeutic strategy requires a large amount of recombinant a-Gal A. The purification strategy present here could be useful for the efficient purification of recombinant enzyme.

Acknowledgments This work was supported in part by Grants-in-Aid from Mizutani Foundation for Glycoscience and American Heart Association (AHA 0130522T).

[15]

[16]

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