CLONING AND EXPRESSION OF BIOLOGICALLY ACTIVE ALPHA-GALACTOSIDASE A

Abstract: The present invention involves the production of large quantities of human alpha-Gal A by cloning and expressing the alpha-Gal A coding sequence in eukaryotic host cell expression systems. The eukaryotic expression systems, and in particular the mammalian host cell expression system described herein provide for the appropriate cotranslational and posttranslational modifications required for proper processing, e.g., glycosylation, phosphorylation, etc. and sorting of the expression product so that an active enzyme is produced. In addition, the expression of fusion proteins which simplify purification is described. Using the methods described herein, the recombinant alpha-Gal A is secreted by the engineered host cells so that it is recovered from the culture medium in good yield. The alpha-Gal A produced in accordance with the invention may be used, but is not limited to, in the treatment in Fabry Disease; for the hydrolysis of alpha-galactosyl residues in glycoconjugates; and/or for the conversion of the blood group B antigen on erythrocytes to the blood group O antigen.

Description: CLONING AND EXPRESSI ON OF BIOLOGICALLY ACTIVE ALPHA-GALΛCTOS IDASE A 1. INTRODUCTION The present invention relates to the production of biologically active human α-Galactosidase (α-Gal A) involving cloning and expression of the genetic coding sequence for α-Gal A in eukaryotic expression systems which provide for proper post-translational modifications and processing of the expression product. The invention is demonstrated herein by working examples in which high levels of α-Gal A were produced in mammalian expression systems. The α-Gal enzyme produced in accordance with the invention may be used for a variety of purposes, including but not limited to enzyme replacement therapy for Fabry Disease, industrial processes involving the hydrolysis of α-D— galactosyl residues of glycoconjugates, and for the conversion of the blood group B antigen on erythrocytes to the blood group O antigen. 2. BACKGROUND OF THE INVENTION In the early 1970's, several investigators demonstrated the existence of two α-Galactosidase isozymes designated A and B, which hydrolyzed the α- galactosidic linkages in 4-MU-and/or p-NP-α-D— galactopyranosides (Kint, 1971, Arch. Int. Physiol. Biochem. 79: 633-644; Beutler & Kuhl, 1972, Amer. J. Hum. Genet. 24: 237-249; Romeo, et al., 1972, FEBS Lett. 27: 161-166; Wood & Nadler, 1972, Am. J. Hum. Genet. 24: 250-255; Ho, et al. , 1972, Am. J. Hum. Genet. 24: 256-266; Desnick, et al., 1973, J. Lab. Clin. Med. 81: 157-171; and Desnick, et al. , 1989, in The Metabolic Basis of Inherited Disease, Scriver, C.R. , Beaudet, A.L. Sly, W.S. and Valle, D. , eds, pp. 1751-1796, McGraw Hill, New York). In tissues, about 80%-90% of total α-Galactosidase (α-Gal) activity was due to a thermolabile, myoinositol-inhibitable α-Gal A isozyme, while a relatively thermostable, α-Gal B, accounted for the remainder. The two "isozymeε" were separable by electrophoresis, isoelectric focusing, and ion exchange chromatography. After neuraminidase treatment, the electrophoretic migrations and pi value of α-Gal A and B were similar (Kint, 1971; Arch. Int. Physiol. Biochem. 79: 633-644), initially suggesting that the two enzymes were the differentially glycosylated products of the same gene. The finding that the purified glycoprotein enzymes had similar physical properties including εubunit molecular weight (-46 kDa) , homodimeric structures, and amino acid compositions also indicated their structural relatedness (Beutler & Kuhl, 1972, J. Biol. Che . 247: 7195-7200; Callahan, et al. , 1973, Biochem. Med. 7: 424-431; Dean, et al., 1977, Biochem. Biophys. Res. Comm. 77: 1411-1417; Schra , et al., 1977, Biochim. Biophys. Acta. 482: 138-144; Kusiak, et al., 1978, J. Biol. Chem. 253: 184-190; Dean, et al. , 1979, J. Biol. Chem. 254: 10001-10005; and Bishop, et al., 1980, in Enzyme Therapy in Genetic Disease: 2, Desnick, R.J. , ed., pp. 17-32, Alan R. Liss, Inc., New York). However, the subsequent demonstration that polyclonal antibodies against α-Gal A or B did not cross-react with the other enzyme (Beutler & Kuhl, 1972, J. Biol. Chem. 247: 7195-7200; and Schram, et al. , 1977, Biochim. Biophys. Acta. 482: 138-144); that only α-Gal A activity was deficient in hemizygotes with Fabry disease (Kint, 1971; Arch. Int. Physiol. Biochem. 79: 633-644; Beutler & Kuhl, 1972, Amer. J. Hum. Genet. 24: 237-249; Romeo, et al., 1972, FEBS Lett. 27: 161- 166; Wood & Nadler, 1972, Am. J. Hum. Genet. 24: 250- 255; Ho, et al., 1972, Am. J. Hum. Genet. 24: 256-266; Desnick, et al., 1973, J. Lab. Clin. Med. 81: 157-171; Desnick, et al., 1989, in The Metabolic Basis of Inherited Disease, Scriver, C.R. , Beaudet, A.L. Sly, W.S. and Valle, D., eds, pp. 1751-1796, McGraw Hill, New York; and, Beutler & Kuhl, 1972, J. Biol. Chem. 247: 7195-7200); and that the genes for α-Gal A and B mapped to different chromosomes (Desnick, et al., 1989, in The Metabolic Basis of Inherited Disease, Scriver, C.R., Beaudet, A.L. Sly, W.S. and Valle, D., eds, pp. 1751-1796, McGraw Hill, New York; deGroot, et al., 1978, Hum. Genet. 44: 305-312), clearly demonstrated that these enzymes were genetically distinct. 2.1. α-GAL A AND FABRY DISEASE In Fabry disease, a lysosomal storage disease resulting from the deficient activity of α-Gal A, identification of the enzymatic defect in 1967 (Brady, et al., 1967, N. Eng. J. Med. 276: 1163) led to the first in vitro (Dawson, et al., 1973, Pediat. Res. 7: 694-690m) and jln vivo (Mapes, et al. , 1970, Science 169: 987) therapeutic trials of α-Gal A replacement in 1969 and 1970, respectively. These and subsequent trials (Mapes, et al., 1970, Science 169: 987; Desnick, et al., 1979, Proc. Natl. Acad. Sci. USA 76: 5326; and, Brady,- et al., 1973, N. Engl. J. Med. 289: 9) demonstrated the biochemical effectiveness of direct enzyme replacement for this disease. Repeated injections of purified splenic and plasma α-Gal A (100,000 U/injection) were administered to affected hemizygotes over a four month period (Desnick, et al., 1979, Proc. Natl. Acad. Sci. USA 76: 5326). The results of these studies demonstrated that (a) the plasma clearance of the splenic form was 7 times faster than that of the plasma form (10 min vs 70 min) ; (b) compared to the splenic form of the enzyme, the plasma form effected a 25-fold greater depletion of plasma substrate over a markedly longer period (48 hours vs l hour) ; (c) there was no evidence of an immunologic response to six doses of either form, administered intravenously over a four month period to two affected hemizygotes; and (d) suggestive evidence was obtained indicating that stored tissue substrate was mobilized into the circulation following depletion by the plasma form, but not by the splenic form of the enzyme. Thus, the administered enzyme not only depleted the substrate from the circulation (a major site of accumulation) , but also possibly mobilized the previously stored substrate from other depots into the circulation for subsequent clearance. These studies indicated the potential for eliminating, or significantly reducing, the pathological glycolipid storage by repeated enzyme replacement. However, the biochemical and clinical effectiveness of enzyme replacement in Fabry disease has not been demonstrated due to the lack of sufficient human enzyme for adequate doses and long- term evaluation. 2 ' 2 - THE α-Gal A ENZYME The α-Gal A human enzyme has a molecular weight of approximately 101,000 Da. On SDS gel electrophoresis it migrates as a single band of approximately 49,000 Da indicating the enzyme is a homodimer (Bishop & Desnick, 1981, J. Biol. Chem. 256: 1307). α-Gal A is synthesized as a 50,500 Da precursor containing phosphorylated endoglycosidase H sensitive oligosaccharides. This precursor is processed to a mature form of about 46,000 Da within 3-7 days after its synthesis. The intermediates of this processing have not been defined (Lemansky, et al., 1987, J. Biol. Chem. 262: 2062) . As with many lysosomal enzymes, α-Gal A is targeted to the lysosome via the mannose-6-phoεphate receptor. This is evidenced by the high secretion rate of this enzyme in mucolipidosis II cells and in fibroblastε treated with NH 4 C1. The enzyme has been shown to contain 5-15% Asn linked carbohydrate (Ledonne, et al., 1983, Arch. Biochem. Biophys. 224: 186). The tissue form of this enzyme was shown to have -52% high mannose and 48% complex type oligosaccharides. The high mannose type coeluted, on Bio-gel chromatography, with Man 8 . 9 GlcNAc while the complex type oligosaccharides were of two categories containing 14 and 19-39 glucose units. Upon isoelectric focusing many forms of this enzyme are observed depending on the sources of the purified enzyme (tissue vs plasma form) . However, upon treatment with neura inidase, a single band is observed (pl~5.1) indicating that this heterogeneity is due to different degrees of sialylation (Bishop & Desnick, 1981, J. Biol. Chem. 256: 1307). Initial efforts to express the full-length cDNA encoding α-Gal A involved using various prokaryotic expression vectors (Hantzopoulos and Calhoun, 1987, Gene 57:159; Ioannou, 1990, Ph.D. Thesis, City University of New York) . Although microbial expression was achieved, as evidenced by enzyme assays of intact E_j_ coli cells and growth on melibiose as the carbon source, the human protein was expressed at low levels and could not be purified from the bacteria. These results indicate that the recombinant enzyme was unstable due to the lack of normal glycosylation and/or the presence of endogenous cytoplasmic or periplasmic proteases. 2.3. LYSOSOMAL ENZYMES: BIOSYNTHESIS AND TARGETING Lysosomal enzymes are synthesized on membrane- bound polysomes in the rough endoplasmic reticulum. Each protein is synthesized as a larger precursor containing a hydrophobic amino terminal signal peptide. This peptide interacts with a signal recognition particle, an IIS ribonucleoprotein, and thereby initiates the vectoral transport of the nascent protein across the endoplasmic reticulum membrane into the lumen (Erickson, et al., 1981, J. Biol. Chem. 256: 11224; Erickson, et al., 1983, Biochem. Biophys. Res. Commun. 115: 275; Rosenfeld, et al., 1982, J. Cell Biol. 93: 135). Lysosomal enzymes are cotranslationaly glycosylated by the en bloc transfer of a large preformed oligosaccharide, glucose-3, mannose-9, N-acetylglucosamine-2, from a lipid-linked intermediate to the Asn residue of a consensus sequence Asn-X-Ser/Thr in the nascent polypeptide (Kornfeld, R. & Kornfeld, S. , 1985, Annu. Rev. Biochem. 54: 631). In the endoplasmic reticulum, the signal peptide is cleaved, and the processing of the Asn-linked oligosaccharide begins by the excision of three glucose residues and one mannose from the oligosaccharide chain. The proteins move via vesicular transport, to the Golgi stack where they undergo a variety of posttranslational modifications, and are sorted for proper targeting to specific destinations: lysosomes, secretion, plasma membrane. During movement through the Golgi, the oligosaccharide chain on secretory and membrane glycoproteins is processed to the sialic acid-containing complex-type. While some of the oligosaccharide chains on lysosomal enzymes undergo similar processing, most undergo a different series of modifications. The most important modification is the acquisition of phosphomannosyl residues which serve as an essential component in the process of targeting these enzymes to the lysosome (Kaplan, et al., 1977, Proc. Natl. Acad. Sci. USA 74: 2026). This recognition marker is generated by the sequential action of two Golgi enzymes. First, N- acetylglucosaminylphosphotransferase transfers N- acetylglucosamine-1-phosphate from the nucleotide sugar uridi e diphoεphate-N-acetylglucosamine to selected mannose residues on lysosomal enzymes to give rise to a phosphodiester intermediate (Reitman & Kornfeld, 1981, J. Biol. Chem. 256: 4275; Waheed, et al., 1982, J. Biol. Chem. 257: 12322). Then, N- acetylglucosamine-1-phosphodiester α-N- acetylglucosaminidase removes N-acetylglucosamine residue to expose the recognition signal, mannose-6- phosphate (Varki & Kornfeld, 1981, J. Biol. Chem. 256: 9937; Waheed, et al. , 1981, J. Biol. Chem. 256: 5717). Following the generation of the phosphomannosyl residues, the lysosomal enzymes bind to mannose-6- phosphate (M-6-P) receptors in the Golgi. In this way the lysosomal enzymes remain intracellular and segregate from the proteins which are destined for secretion. The ligand-receptor complex then exits the Golgi via a coated vesicle and is delivered to a prelysosomal staging area where dissociation of the ligand occurs by acidification of the compartment (Gonzalez-Noriega, et al. , 1980, J. Cell Biol. 85: 839) . The receptor recycles back to the Golgi while the lysosomal enzymes are packaged into vesicles to form primary lysosomes. Approximately, 5-20% of the lysosoml enzymes do not traffic to the lysosomes and are secreted presumably, by default. A portion of these secreted enzymes may be recaptured by the M-6-P receptor found on the cell surface and be internalized and delivered to the lysosomes (Willingham, et al. , 1981, Proc. Natl. Acad. Sci. USA 78: 6967). Two mannose-6-phosphate receptors have been identified. A 215 kDa glycoprotein has been purified from a variety of tissues (Sahagian, et al., 1981, Proc. Natl. Acad. Sci. USA, 78: 4289; Steiner & Rome, 1982, Arch. Biochem. Biophys. 214: 681). The binding of this receptor is divalent cation independent. A second M-6-P receptor also has been isolated which differs from the 215 kd receptor in that it has a requirement for divalent cations. Therefore, this receptor is called the cation-dependent (M-β-P 00 ) while the 215 kd one is called cation-independent (M-6-P α ) . The M-ό-P 00 receptor appears to be an oligomer with three subunits with a subunit molecular weight of 46 kDa. 3. SUMMARY OF THE INVENTION The present invention involves the production of large quantities of human α-Gal A by cloning and expressing the α-Gal A coding sequence in eukaryotic host cell expression systems. The eukaryotic expression systems, and in particular the mammalian host cell expression system described herein, provide for the appropriate cotranslational and posttranslational modifications required for proper processing, e.g. , glycosylation, sialylation, phosphorylation, etc. and sorting of the expression product so that an active enzyme is produced. Also described.is the expression of α-galactosidase A fusion proteins which are readily purified. These fusion proteins are engineered so that the α- galactosidase A moiety is readily cleaved from the fusion protein and recovered. Using the methods described herein, the recombinant α-Gal A is secreted by the engineered host cells so that it is recovered from the culture medium in good yield. The α-Gal A produced in accordance with the invention may be used for a variety of ends, including but not limited to the treatment in Fabry Disease, the conversion of blood type B to O, or in any commercial process that involves the hydrolysis of α-D—galactosyl residues from glycoconjugates. Further, this invention describes a method whereby proteins that are normally intracellularly targeted may be overexpressed and secreted from recombinant mammalian cell lines. 3.1. DEFINITIONS As used herein, the following terms and abbreviations will have the indicated meaning: α-Galactosidase A α-Gal A α-N-AcetylGalactosaminidase α-GalNAc base pair(s) bp Chinese hamster ovary CHO complementary DNA CDNA counts per minute cpm deoxyribonucleic acid DNA Dulbecco's Modified Eagle's Medium DMEM fetal calf serum FCS kilobase pairs kb kilodalton kDa mannose-6-phosphate M-6-P methotrexate MTX 4-methylumbelliferyl-α-D—galactoside 4-MU-α-Gal 4-methyl-umbelliferyl-α-N-acetyl- galactosaminide 4-Mu-α-GalNAc micrograms micrometer μm nanograms ng nanometer nm nucleotide nt p-nitrophenyl-α-N-Acetylgalactosaminide pNP-α-GalNAc polyacrylamide gel electrophoresis PAGE polymerase chain reaction PCR ribonucleic acid RNA sodium dodecyl sulfate SDS units U 4 - DESCRIPTION OF THE FIGURES FIG. lA-lC. Full-length human α-Gal cDNA sequence. N-terminal, cyanogen bromide (CB) , and tryptic (T) peptide amino acid sequences obtained from peptide microsequencing are indicated by underlines. Differences from the sequence predicted from the cDNA are shown. The four putative N-glycosylation sites are denoted and the 3 ' termination signals are overlined. FIG. 1D-1F. Alignment of amino acid sequences deduced from the full-length cDNAs encoding human α- Gal-NAc (α-Gal B) , α-Gal, yeast Mel 1, and E____ coli Mel A. Colons, identical residues; single dots, isofunctional amino acids; and boxes, identical residues in α-GalNAc, α-Gal, Mel 1 and/or Mel A. Gaps were introduced for optimal alignment. Numbered vertical lines indicate exon boundaries for α-Gal (Bishop, et al, 1988, Proc. Natl. Acad. Sci. USA 85: 3903-3907) . FIG. 1G. Construction of the α-Gal mammalian expression vector p91-AGA. The full-length cDNA was excised from plasmid pcDAG126, adapted by the addition of Eco RI linkers and subsequently cloned into the Eco RI site of expression vector p91023(B) . FIG. 2A-2B. Transient expression of human α-Gal in COS-1 cells. Maximum activity (U/mg) was reached 72 hours post-transfection in cells receiving the p91-AGA construct. No increase in α-Gal activity was observed in cells receiving no plasmid DNA nor in cells receiving the p91 vector with the α-Gal cDNA in the reverse orientation. FIG. 3A-3B. Serum effect on secretion of recomb¬ inant α-Gal by CHO DG5.3. Cells were plated in DMEM supplemented with the appropriate serum con-centration (FIG. 3A. Cells were plated in DMEM sup-plemented with 10% FCS. Following confluency ( ~ 4 days) , the media was replaced with fresh DMEM sup-plemented with the appropriate serum concentration (FIG. 3B) . FIG. 4. High-level production of recombinant α- Gal in a hollow fiber bioreactor. The amount of fetal bovine serum required by this system for optimal cell- growth and protein secretion could be decreased to about 1%. FIG. 5. SDS-PAGE of each step of the α-Gal purificiation scheme. Lanes 1, 6, molecular weight markers; lane 2, crude media; lane 3, affinity chromatography; lane 4, octyl-Sepharose chromatography; lane 5, superose 6 chromatography. FIG. 6. Total cellular (lanes 1-4) and media (lanes 5-8) from control DG44 cells (lane 1,5) , DG5 cells (lanes 2 , 6) , DG5.3 cells (lanes 3,7) and DG11 cells (lanes 4,8), labeled with [ 35 S]-methionine. FIG. 7A-7C. Physicokinetic properties of recombinant α-Gal . Km towards the artificial substrate 4-MU-α-D—galactopyranoside (FIG. 7A) . Isoelectric point of recombinant and human plasma purified enzyme (FIG. 7B) . pH optimum of the recombinant enzyme. (FIG. 7C) . FIG. 8A-8B. P-C 12 STH degradation by CHO DG5.3 cells overproducing human α-Gal. Rapid degradation of this substrate is observed by the accumulation of P-C 12 SDH. FIG. 9. Acquisition of disulfide bridges by recombinant α-Gal . CHO DG5.3 cells were labeled with [ 35 S]-methionine and chased for the indicated times. SDS-PAGE in the absence of a reducing agent reveals the formation of secondary structures through disulfide bond formation. FIG. 10. Arrival of newly synthesized α-Gal to the Golgi network detected by the acquisition of Endo H resistent oligosaccharides. FIG. 11. Secretion rate of recombinant α-Gal . CHO DG5.3 cells were labeled with [ 3S S]-methionine for 5 min and chased with cold methionine. Culture media aliquots were removed at the indicated times and immunoprecipitated with anti-α-Gal polyclonal antibodies. FIG. 12. SDS-PAGE of culture media from DG44 (lane 1; control), DG5 (lane 2) and DG5.3 (lanes 3,4) cells labeled with [ 35 S]-methionine for 1 hour (lanes -3 ) and 24 hours (lane 4) . FIG. 13. Analysis of the carbohydrate moieties on recombinant α-Gal . CHO DG5.3 cells were labeled with [ 35 S]-methionine for 24 hours, the culture media collected and the recombinant enzyme immunoprecipitated. Aliquots were digested with endo D (lane 2), Endo H (lane 3), Endo F (lane 4), PNGase F (lane 5) , Endo D and H (lane 6) , Endo H and F (lane 7), and Endo H, F, and PNGase F (lane 8). Untreated samples (lanes 1, 9) . FIG. 14. Cellular (lanes 1,3) and secreted (lanes 2,4) forms of recombinant α-Gal treated with PNGase F (lanes 3,4). Controls (lanes 1,2). FIG. 15. Effect of glycosylation inhibitors on the secretion of recombinant α-Gal. FIG. 16. 32 P labelling of CHO DG44 (lanes 2, 3) and DG5.3 (lanes l, 4) . α-Gal was immunoprecipitated from cells (lanes 1 , 2 ) and media (lanes 2 , 3 ) . FIG. 17A-17D. QAE-Sephadex chromatography of endo H sensitive oligosaccharides of recombinant α- Gal. Untreated, dilute HCl treated, neuraminidase treated and alkaline phosphatase treated oligosaccharides. FIG. 18. Endo H sensitive oligosaccharides of recombinant α-Gal chromatographed on M-6-P receptor, solid circles, peak minus 4, open circles, peak minus 2. FIG. 19. Recombinant α-Gal chromatography on M- 6-P receptor. DG5.3 cells labeled with [ 35 S]- methionine for 24 hours and media collected for chromatography. Solid circles, α-Gal activity; open boxes, total radioactivity. FIG. 20A-20C. Recombinant and human α-Gal affinity chromatography on M-6-P receptor. Cells were labeled with [ 35 S]-methionine for 24 hours in the presence of NH 4 C1 and the culture media were collected. DG5.3 secretions (FIG. 20A) , MS914 secretions (FIG. 20B) and 293 secretions (FIG. 20C) . Solid circles, α- Gal activity. Squares, total radioactivity. Open circles, M-6-P gradient used for elution. FIG. 21. Uptake of recombinant α-Gal by Fabry fibroblasts. Cells were incubated for the indicated amounts of α-Gal for 6 hours. Open circles, α-Gal uptake, closed circles, uptake in the presence of 2mM M-6-P. FIG. 22. Construction scheme of the α-Gal protein A fusion. The fusion was accomplished in two separate PCR reactions as described in Section 9.1. FIG. 23. Nucleotide sequence of the protein A domain E, collagenase cleavage sequence and 3'α-Gal RATIFIED SHEET (RULE 91) sequence (23A) . Schematic of the fusion construct showing the collagenase consensus in relation to the α-Gal and protein A domains (23B) . FIG. 24A-24B. Plasma clearance following intravenous administration to mice of the recombinant human secreted α-galactosidase A containing α2,6- sialic acid residues (Δ—Δ) and the non-α2,6- sialylated glycoform (•—•) upper graph, compared with the plasma clearance of these glycoforms following treatment with acid phosphatase. Each point represents the average of the values from two independent injections. The T, /: values were estimated by extrapolation. FIG. 25. Tissue distribution of different forms of recombinant human secreted α-Gal A following intravenous administration to mice. Each point represents the average of the values from two independent injections. FIG. 26. DG5.3-1000MX CHO Cells Contain Crystalline Structures of Overexpressed Human α-Gal A. Electron micrographs of DG5.3-1000MX cells showing crystalline structures in single membrane-limited vacuoles (26A) and in vesicles, presumably in the dilated trans-Golgi (26B; bar, 0.15 μm and 0.10 μm, respectively) . 26C and D: Immunoelectron microscopic localization of human α-Gal A with 10 nm colloidal gold particles (bar, 0.31 μm snf 0.19 μm, respectively) . 26E: Electron micrograph of parental dfhr DG44 cells (bar, 1.11 μm) ; inset showing Golgi complex (arrows) in dfhr DG44 cells (bar, 0.5 μm) . FIG. 27A-27C. Aggregation of Purified Secreted α-Gal A is Enzyme Concentration and pH Dependent. 27A: Precipitation of α-Gal A (10 mg/ml) with decreasing pH. Note that about 12% of incubated enzyme was precipitated at pH 6.0, the estimated pH of the trans-Golgi Network (TGN) . 27B: Turbidity of increasing concentrations of secreted α-Gal A at pH 5.0 (closed circles) and pH 7.0 (closed triangles) in the absence of Bovine Serum Albumin (BSA) . As a control for non-specific precipitation with increasing protein concentration, secreted α-Gal A (1 mg/ml) was mixed with increasing BSA concentrations (0.1 to 10 mg/ml) at pH 5.0 (solid squares). 27C: SDS-PAGE of the supernatant and pellet fractions from mixtures of purified secreted α-Gal A (10 mg/ml) and BSA (2 mg/ml) incubated at decreasing pH values. Note that α-Gal A was precipitated with decreasing pH whereas the concentrations of soluble and precipitated BSA were essentially unchanged. FIG. 28. Aggregation-Secretion Model for Selective Secretion of Human α-Gal A Overexpressed in CHO Cells. High level overexpression in CHO cells of human α-Gal A or other lysosomal enzymes normally targeted to lysosomes results in their selective secretion due to their aggregation and the resultant inaccessibility of their M6PR signals. The enzymes undergo normal post-translational processing until arriving in the TGN, where the overexpressed enzyme undergoes protein-protein interactions and forms smaller soluble and larger particulate aggregates, due to lower pH of the TGN. The TGN becomes dilated with the overexpressed enzyme. Some aggregates and soluble enzyme with exposed M6P signals are trafficked to lys- osomes, while the majority of aggregates whose M6P are not accessible are exocytosed by default via the constitutive secretory pathway. Also, decreased sulfation may occur as the tyrosines in enzyme aggr¬ egates destined for secretion are unavailable to sulf- otransferase. The model may explain the selective secretion of other - - other overexpressed proteins that normally are targeted to specific organelles. 5. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the production of biologically active human α-Gal A involving cloning and expressing the nucleotide coding sequences for the enzyme in eukaryotic expression systems. Successful expression and production of this purified, biologically active enzyme as described and exemplified herein is particularly significant for a number of reasons. For example, past efforts to express the full-length cDNA encoding α-Gal A using various prokaryotic expression vectors resulted in expression of the enzyme, as evidenced by enzyme assays of intact microbial host cells and growth on melibiose as the carbon source; however, the human enzyme was expressed at low levels and could not be purified from the bacteria. These results indicate that the recombinant enzyme expressed in microbial systems was unstable due to the lack of normal glycosylation and/or the presence of endogenous cytoplasmic or periplasmic proteases. Efforts to express this enzyme in eukaryotic expression systems were equally difficult for different reasons. The α-Gal A is a lysosomal enzyme encoded by a "housekeeping" gene. The primary translation product is highly modified and processed, requiring a complex series of events involving cleavage of a signal sequence, glycosylation, phosphorylation, and sialylation, which can be properly effected only by appropriate host cells. Moreover, since the expression product is destined for the lysosome, which remains intracellular, it is quite surprising that the methods described herein allow for the secretion of a properly processed, biologically active molecule. The biologically active α-Gal A produced in accordance with the invention has a variety of uses, probably the most significant being its use in enzyme replacement therapy for the lysosomal storage disorder, Fabry disease. For example, the metabolic defect in cultured fibroblasts from Fabry disease can be corrected in vitro by the addition of exogenous α- Gal A into the culture medium. In addition, limited human trials have demonstrated the biochemical effectiveness of enzyme replacement to deplete the circulating substrate prior to vascular deposition. However, prior to the present invention, large quantities of biologically active, purified human a- Gal A could not be produced for use in replacement therapies. The α-Gal A produced in accordance with the invention also has a number of industrial uses, e.g.. in any process involving the hydrolysis of α-D— galactoεyl glycoconjugates, the conversion of blood group B to group O, etc. , as described herein. The invention is divided into the following sections solely for the purpose of description: (a) the coding sequence for α-Gal A; (b) construction of an expression vector which will direct the expression of the enzyme coding sequence; (c) transfection of appropriate host cells which are capable of replicating, translating and properly processing the primary transcripts in order to express a biologically active gene product; and (d) identification and/or purification of the enzyme so produced. Once a transformant is identified that expresses high levels of biologically active enzyme, the practice of the invention involves the expansion and use of that clone in the production and purification of biologically active α-Gal A. The invention is demonstrated herein, by way of examples in which cDNAs of α-Gal A were cloned and expressed in a mammalian expression system. Modifications to the cDNA coding sequences which improve yield, and simplify purification without detracting from biological activity are also described. Further, modifications to the host cells are described that allow for the expression of sialylated and asialylated glycoforms of the enzyme, both of which may be easily purified. Although the invention is described for α-Gal A, the methods and modifications explained may be analogously applied to the expression of other secreted proteins, and in particular, other lysosomal enzymes, including but not limited to α-N-acetylgalactosaminidase, and acid sphingomyelinase. Various aspects of the invention are described in more detail in the subsections below and in the examples that follow. 5.1. THE α-GAL A CODING SEQUENCE The nucleotide coding sequence and deduced amino acid sequence for α-Gal A is depicted in FIG. 1A-1C. This nucleotide sequence, or fragments or functional equivalents thereof, may be used to generate recombinant DNA molecules that direct the expression of the enzyme product, or functionally active peptideε or functional equivalents thereof, in appropriate host cells. Due to the degeneracy of the nucleotide coding sequence, other DNA sequences which encode sub- stantially the same amino acid sequences as depicted in FIG. 1A-1C may be used in the practice of the invention for the cloning and expression of α-Gal A. Such alterations include deletions, additions or substitutions of different nucleotide residues resulting in a sequence that encodes the same or a functionally equivalent gene product. The gene product may contain deletions, additions or substitutionε of amino acid reεidueε within the sequence, which result in a silent change thus producing a bioactive product. Such amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, the amphipathic nature of the residues involved and/or on the basis of crystallographic data. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; amino acids with uncharged polar head groups having similar hydrophilicity values include the following: leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine; serine, threonine; phenylalanine, tyrosine. The coding sequences for α-Gal A may be conveniently obtained from genetically engineered microorganisms or cell lines containing the enzyme coding sequences, such as the deposited embodiments described herein. Alternatively, genomic sequences or cDNA coding sequences for these enzymes may be obtained from human genomic or cDNA libraries. Either genomic or cDNA libraries may be prepared from DNA fragments generated from human cell sources. The fragments which encode α-Gal A may be identified by screening such libraries with a nucleotide probe that is substantially complementary to any portion of the sequence depicted in FIG. 1A-1C. Indeed, sequences generated by polymerase chain reaction can be ligated to form the full-length sequence. Although portions of the coding sequences may be utilized, full length clones, i.e. , those containing the entire coding region for α-Gal A, may be preferable for expression. Alternatively, the coding sequences depicted in FIG. 1A-1C may be altered by the addition of sequences that can be used to increase levels of expression and/or to facilitate purification. For example, as demonstrated in the working embodiments described herein, the α-Gal A coding sequence was modified by the addition of the nucleotide sequence encoding the cleavage site for collagenase followed by the Staphylococcal Protein A. Exression of this chimeric gene construct resulted in a fusion protein consisting of α-Gal A- the collagenase substrate — Protein A. This fusion protein was readily purified using an IgG column which binds to the Protein A moiety. Unfused α-Gal A was released from the column by treatment with collagenase which cleaved the α-Gal A from the Protein A moiety bound to the column. Other enzyme cleavage substrates and binding proteins can be engineered into similar constructs for the production of α-Gal A which can be readily purified and released in its biologically active form. Techniques well-known to those skilled in the art for the isolation of DNA, generation of appropriate restriction fragments, construction of clones and libraries, and screening recombinants may be used. For a review of such techniques, see, for example, Sambrook, et al., 1989, Molecular Cloning A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, N.Y., Chapters 1-18. In an alternate embodiment of the invention, the coding sequence of FIG. 1A-1C could be synthesized in whole or in part, using chemical methods well-known in the art. See, for example, Caruthers, et. al. , 1980, Nuc. Acids Res. Symp. Ser. 7: 215-233; Crea & Horn, 1980, Nuc. Acids Res. 9(10): 2331; Matteucchi & Carruthers, 1980, Tetrahedron Letters 21: 719; and Chow and Kempe, 1981, Nuc. Acids Res. 9(12): 2807- 2817. Alternatively, the protein itself could be produced using chemical methods to synthesize the amino acid sequence depicted in FIG. 1A-1C in whole or in part. For example, peptides can be synthesized by solid phase techniques, cleaved from the resin and purified by preparative high performance liquid chromatograph. (E.g. , see, Creighton, 1983, Proteins, Structures and Molecular Principles, W.H. Freeman & Co., N.Y. pp. 50-60). The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g.. the Edman degradation procedure; see Creighton, 1983, Proteins, Structures and Molecular Principles, W. H. Freeman & Co., N.Y., PP- 34-49) . Human α-Gal A is a homodimeric glycoprotein. The full-length α-Gal A cDNA predicts a mature subunit of 398 amino acids. The amino acid sequence has an overall homology of about 50% with human α-N- acetylgalactosaminidase (α-Gal B) . Homology searches with computerized data baseε revealed short regions of α-Gal A homology with the yeast Mel 1 and the E. coli Mel A amino acid sequences (see FIG. 1D-1F) . It is likely that these conserved regions are important for enzyme conformation, stability, subunit association and/or catalysiε. Thus, it is preferred not to alter such conserved regions. However, certain modifications in the coding sequence may be advantageous. For example, the four N-linked glycosylation consensus sequences could be selectively obliterated, thereby altering the glycosylation of the enzyme and affecting phosphorylation, εialylation, εulfation, etc. Such modified enzymes may have altered clearance properties and targeting when injected into Fabry patients. Oligosaccharide modifications may be useful in the targeting of α-Gal A for effective enzyme therapy. Some examples of such modifications are described in more detail infra. Previous studies demonstrated that the plasma glycoform of α-Gal A, which is more highly sialylated than the splenic glycoform, was more effective in depleting the toxic accumulated circulating substrate from Fabry patients (Desnick et al., 1977, Proc. Natl. Acad. Sci. USA 76:5326-5330). Studies characterizing the purified splenic and plasma glycoforms of the enzyme revealed differences only in their oligosaccharide moieties (Desnick et al., 1977, Proc. Natl. Acad. Sci. USA 76:5326-5330). Thus, efforts to target the recombinant enzyme for effective treatment of Fabry disease may be enhanced by modification of the N-glycosylation sites. Also, the 5' untranslated and coding regions of the nucleotide sequence could be altered to improve the translational efficiency of the α-Gal A mRNA. For example, substitution of a cytosine for the guanosine in position +4 of the α-Gal A cDNA could improve the translational efficiency of the α-Gal A mRNA 5- to 10- fold (Kozak, 1987, J. Mol. Biol. 196:947-950). In addition, based on X-ray crystallographic data, sequence alterations could be undertaken to improve protein stability, e.g.. introducing disulfide bridges at the appropriate positions, and/or deleting or replacing amino acids that are predicted to cause protein instability. These are only examples of modifications that can be engineered into the α-Gal A enzyme to produce a more active or stable protein, more enzyme protein, or even change the catalytic specificity of the enzyme. 5.2. PRODUCTION OF RECOMBINANT α-Gal A In order to express a biologically active α-Gal A, the coding sequence for the enzyme, a functional equivalent, or a modified sequence, as described in Section 5.1., supra. is inserted into an appropriate eukaryotic expression vector, i.e.. a vector which contains the necessary elements for transcription and translation of the inserted coding sequence in appropriate eukaryotic host cells which possess the cellular machinery and elements for the proper processing, i.e.. signal cleavage, glycosylation, phosphorylation, sialylation, and protein sorting. Mammalian host cell expression systems are preferred for the expression of biologically active enzymes that are properly folded and processed; when administered in humans such expression products should exhibit proper tissue targeting and no adverse imraunological reaction. 5.2.1. CONSTRUCTION OF EXPRESSION VECTORS AND PREPARATION OF TRANSFECTANTS Methods which are well-known to those skilled in the art can be used to construct expression vectors containing the α-Gal A coding sequence and appropriate transcriptional/translational control signals. These methods include n vitro recombination/genetic recombination. See, for example, the techniques described in Maniatis et al., 1982, Molecular Cloning A Laboratory Manual, Cold spring Harbor Laboratory, N.Y. , Chapter 12. A variety of eukaryotic host-expression systems may be utilized to express the α-Gal A coding sequence. Although prokaryotic systems offer the distinct advantage of ease of manipulation and low cost of scale-up, their major drawback in the expression of α-Gal A is their lack of proper post- translational modifications of expressed mammalian proteins. Eukaryotic systems, and preferably mammalian expression systems, allow for proper modification to occur. Eukaryotic cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, phosphorylation, and, advantageously secretion of the gene product should be used as host cells for the expression of α-Gal A. Mammalian cell lines are preferred. Such host cell lines may include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, -293, WI38, etc. Alternatively, eukaryotic host cells which possess some but not all of the cellular machinery required for optional processing of the primary transcript, and/or post-translational processing and/or secretion of the gene product may be modified to enhance the host cell's processing capabilities. For example, a recombinant nucleotide sequence encoding a peptide product that performs a processing function the host cell had not previously been capable of may be engineered into the host cell line. Such a sequence may either be co-transfected into the host cell along with the gene of interest, or included in the recombinant construct encoding the gene of interest. Alternatively, cell lines containing this sequence may be produced which are then transfected with the gene of interest. Appropriate eukaryotic expression vectors should be utilized to direct the expression of α-Gal A in the host cell chosen. For example, at least two basic approaches may be followed for the design of vectors on SV40. The first is to replace the SV40 early region with the gene of interest while the second is to replace the late region (Hammarskjold, et al., 1986, Gene 43: 41). Early and late region replacement vectorε can also be complemented n vitro by the appropriate SV40 mutant lacking the early or late region. Such complementation will produce recombinants which are packaged into infectious capsids and which contain the α-Gal A gene. A permisεive cell line can then be infected to produce the recombinant protein. SV40-based vectors can also be used in transient expression studies, where best results are obtained when they are introduced into COS (CV-1, origin of SV40) cells, a derivative of CV-1 (green monkey kidney cells) which contain a single copy of an origin defective SV40 genome integrated into the chromosome. These cells actively synthesize large T antigen (SV40) , thus initiating replication from any plasmid containing an SV40 origin of replication. In addition to SV40, almost every molecularly cloned virus or retrovirus may used as a cloning or expresεion vehicle. Viral vectorε baεed on a number of retroviruεes (avian and murine) , adenoviruses, vaccinia viruε (Cochran, et al., 1985, Proc. Natl. Acad. Sci. USA 82: 19) and polyoma viruε may be used for expression. Other cloned viruses, such as JC (Howley, et al., 1980, J. Virol 36: 878), BK and the human papilloma viruseε (Heilman, et al., 1980, J. Virol 36: 395) , offer the potential of being used as eukaryotic expression vectors. For example, when using adenovirus expression vectors the α-Gal A coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g. , the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g. , region El or E3) will result in a recombinant virus that is viable and capable of expresεing the human enzyme in infected hoεts (e.g. , see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. (USA) 81: 3655-3659). lternatively, the vaccinia virus 7.5K promoter may be used. (e.g. , see, Mackett et al., 1982, Proc. Natl. Acad. Sci. (USA) 79: 7415-7419; Mackett et al. , 1984, J. Virol. 49: 857-864; Panicali et al., 1982, Proc. Natl. Acad. Sci. 79: 4927-4931). Of particular interest are vectors based on bovine papilloma virus (Sarver, et al., 1981, Mol. Cell. Biol. 1: 486) . These vectors have the ability to replicate as extrachromosomal elements. Shortly after entry of this DNA into mouse cells, the plasmid replicates to about 100 to 200 copies per cell. Transcription of the inserted cDNA does not require integration of the plasmid into the host'ε chromosome, thereby yielding a high level of expreεεion. These vectorε can be uεed for stable expression by including a selectable marker in the plasmid, εuch aε the neo gene. High level expression may also be achieved using inducible promoters such as the metallothionine IIA promoter, heat shock promoters, etc. For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, following the introduction of foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with the α-Gal A or DNA controlled by appropriate expresεion control elementε (e.g. r promoter, enhancer, εequenceε, transcription terminators, polyadenylation sites, etc.), and a selectable marker. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomeε and grow to form foci which in turn can be cloned and expanded into cell lineε. A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell 11: 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48: 2026), and adenine phosphoribosyltransferase (Lowy, et al., 1980, Cell 22: 817) genes can be employed in tk", hgprt " or aprt " cells respectively. Also, antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, et al., 1980, Natl. Acad. Sci. USA 77: 3567; O'Hare, et al., 1981, Proc. Natl. Acad. Sci. USA 78: 1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78: 2072; neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al., 1981, J. Mol. Biol. 150: 1) ; and hygro, which conferε reεistance to hygromycin (Santerre, et al., 1984, Gene 30: 147) genes. Recently, additional selectable genes have been described, namely trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman S Mulligan, 1988, Proc. Natl. Acad. Sci. USA 85: 8047); and ODC (ornithine decarboxylase) which confers resiεtance to the ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue L. , 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.). Alternative eukaryotic expression systems which may be used to express the α-Gal A enzymes are yeast transformed with recombinant yeast expression vectors containing the α-Gal A coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g.. baculovirus) containing the α-Gal A coding sequence; or plant cell systemε infected with recombinant viruε expreεεion vectorε (e.g.. cauliflower moεaic viruε, CaMV; tobacco moεaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g. , Ti plasmid) containing the α-Gal A coding sequence. In yeast, a number of vectors containing constitutive or inducible promoters may be used. For a review see, Current Protocols in Molecular Biology, Vol. 2, 1988, Ed. Ausubel et al. , Greene Publish. Assoc. & Wiley Interscience, Ch. 13; Grant et al., 1987, Expression and Secretion Vectorε for Yeaεt, in Methods in Enzymology, Eds. Wu & Grossman, 31987, Acad. Presε, N.Y., Vol. 153, pp.516-544; Glover, 1986, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch. 3; and Bitter, 1987, Heterologouε Gene Expression in Yeaεt, Methodε in Enzymology, Eds. Berger & Kimmel, Acad. Preεs, N.Y., Vol. 152, pp. 673-684; and The Molecular Biology of the Yeast Saccharo yces, 1982, Eds. Strathern et al.. Cold Spring Harbor Presε, Volε. I and II. For complementation aεεayε in yeaεt, cDNAε for α-Gal A may be cloned into yeaεt episomal plasmids (YEp) which replicate autonomously in yeast due to the presence of the yeaεt 2μ circle. The cDNA may be cloned behind either a constitutive yeaεt promoter such as ADH or LEU2 or an inducible promoter such as GAL (Cloning in Yeast, Chpt. 3, R. Rothstein In: DNA Cloning Vol.11, A Practical Approach, Ed. DM Glover, 1986, IRL Press, Wash., D.C.). Constructs may contain the 5' and 3' non-translated regions of the cognate α- Gal A mRNA or those corresponding to a yeast gene. YEp plas idε transform at high efficiency and the 5 plasmidε are extremely εtable. Alternatively, vectorε may be uεed which promote integration of foreign DNA εequenceε into the yeaεt chromosome. In cases where plant expresεion vectors are used, the expression of the α-Gal A coding sequence may be 0 driven by any of a number of promoters. For example, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson et al., 1984, Nature 310:511-514), or the coat protein promoter of TMV (Takamatεu et al., 1987, EMBO J. 6:307-311) may be 5 used; alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi et al., 1984, EMBO J. 3:1671-1680; Broglie et al. , 1984, Science 224:838- 843); or heat shock promoters, e.g.. soybean hspl7.5-E or hspl7.3-B (Gurley et al., 1986, Mol. Cell. Biol. o 6:559-565) may be used. These conεtructs can be introduced into plant cells uεing Ti plaεmidε, Ri plaεmidε, plant viruε vectorε; direct DNA transformation; microinjection, electroporation, etc. For reviews of such techniques see, for example, 5 Weissbach & Weissbach, 1988, Methodε for Plant Molecular Biology, Academic Press, NY, Section VIII, pp. 421-463; and Grierson & Corey, 1988, Plant Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9. An alternative expresεion εyεtem which could be 0 used to express α-Gal A is an insect system. In one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to expresε foreign genes. The virus grows in Spodoptera frugiperda cells. The α-Gal A sequence may be cloned 5 into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter) . Succesεful inεertion of the coding εequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e. , virus lacking the proteinaceous coat coded for by the polyhedrin gene) . These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed. (E.g.. see Smith et al., 1983, J. Viol. 46:584; Smith, U.S. Patent No. 4,215,051) . 5.2.2. IDENTIFICATION OF TRANSFECTANTS OR TRANSFORMANTS EXPRESSING THE α-Gal A PRODUCT The host cells which contain the α-Gal A coding sequence and which express the biologically active gene product may be identified by at least four general approaches: (a) DNA-DNA or DNA-RNA hybridization; (b) the preεence or abεence of "marker" gene functionε; (c) assessing the level of transcription as measured by the expression of α-Gal A mRNA transcriptε in the hoεt cell; and (d) detection of the gene product as measured by immunoassay or by its biological activity. In the first approach, the presence of the α-Gal A coding sequence inserted in the expresεion vector can be detected by DNA-DNA or DNA-RNA hybridization uεing probes comprising nucleotide sequences that are homologous to the α-Gal A coding sequence substantially as shown in FIG. 1A-1C, or portions or derivatives thereof. In the second approach, the recombinant expression vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g. , thymidine kinase activity, resistance to antibioticε, reεistance to methotrexate, transformation phenotype, occlusion body formation in baculovirus, etc.) . For example, if the α-Gal A coding sequence is inserted within a marker gene sequence of the vector, reco binants containing the α-Gal A coding sequence can be identified by the absence of the marker gene function. Alternatively, a marker gene can be placed in tandem with the α-Gal A sequence under the control of the same or different promoter used to control the expression of the α-Gal A coding sequence. Expression of the marker in response to induction or selection indicates expresεion of the α-Gal A coding εequence. In the third approach, transcriptional activity for the α-Gal A coding region can be assessed by hybridization assays. For example, RNA can be isolated and analyzed by Northern blot using a probe homologous to the α-Gal A coding εequence or particular portions thereof subεtantially aε εhown in FIG. 1A-1C. Alternatively, total nucleic acidε of the hoεt cell may be extracted and assayed for hybridization to εuch probeε. In the fourth approach, the expreεεion of the - Gal A protein product can be assessed immunologically, for example by Western blots, immunoassays such as radioimmuno-precipitation, enzyme-linked immunoassays and the like. The ultimate test of the success of the expresεion system, however, involves the detection of the biologically active α-Gal A gene product. Where the host cell secretes the gene product, the cell free media obtained from the cultured transfectant hoεt cell may be assayed for α-Gal A activity. Where the gene product is not secreted, cell lysates may be assayed for such activity. In either case, a number of assayε can be uεed to detect α-Gal A activity including but not limited to: (a) aεεays employing the synthetic fluorogenic or chromogenic α-D- galactosides such as 4-methylumbelliferyl-α-D— galactopyranoside (Desnick et al., 1973, J. Lab. Clin. Invest. 81:157); (b) assays employing the radiolabeled or fluorescent labeled natural subεtrates such as tritiated globotriaosyl ceramide or pyrene- dodecanoyl-sphingosine-trihexoside (Biεhop and Deεnick, 1981, J. Biol. Chem. 256:1307); and (c) aεεays employing X-α-gal. 5.2.3. PURIFICATION OF THE α-GAL A GENE PRODUCT Once a clone that produceε high levels of biologically active α-Gal A is identified, the clone may be expanded and used to produce large amounts of the enzyme which may be purified using techniques well-known in the art including, but not limited to immunoaffinity purification, chromatographic methods including high performance liquid chromatography and "the like. Where the enzyme is secreted by the cultured cells, α-Gal A may be readily recovered from the culture medium. As demonstrated in the working examples described infra, recombinant α-Gal A was purified from the crude media by affinity chromatography on α-GalNH 2 -Cι 2 - Sepharoεe followed by hydrophobic chromatography on Octyl Sepharoεe and gel filtration on a 100 cm Superoεe 6 column. The recombinant enzyme was essentially homogeneous following the gel filtration step and was >98% pure aε judged by SDS-PAGE. Human recombinant α-Gal A was purified to homogeneity from the media of the CHO cell line, DG5.3, which was εhown to εecrete moεt of the recombinant enzyme. The culture media from this clone was highly enriched for α-Gal A when serum-free medium waε used, constituting greater than 95% of the total extracellular protein. Thus, purification to homogeneity could be accomplished in only three chromatographic steps. Over half a gram of enzyme was produced in three months and from a portion of this, 280 mg was purified with a yield of 80% using only laboratory-scale equipment. Notably, the recombinant enzyme had full enzymatic activity with a specific activity equal to that of the previously purified human enzyme (Bishop, et al., 1978, Biochim. Biophys. Acta. 525: 399; Bishop and Desnick, 1981, J. Biol. Chem. 256:1307). The recombinant enzyme was able to recognize and effectively cleave an analog of the natural substrate, globotriaosylceramide. Where the α-Gal A coding sequence is engineered to encode a cleavable fusion protein, the purification of α-Gal A may be readily accomplished using affinity purification techniqueε. In the working examples described infra, a collagenase cleavage recognition consensus sequence was engineered between the carboxy terminus of α-Gal A and protein A. The resulting fusion protein was readily purified uεing an IgG column that bound the protein A moiety. Unfuεed α-Gal A was readily released from the column by treatment with collagenase. In particular, the overlap extension method (Ho, et al., 1989, Gene 77: 51; Kadowaki, et al., 1989, Gene 76: 161) was used to fuse the full-length α-Gal A cDNA to the protein A domain E of Staphylococcus aureus. Following transfection by electroporation, the α-Gal A activity in COS-l cell extracts was increased 6 to 7-fold. In addition, the transfected cells secreted significant amounts of the fusion protein into the culture media (400 U/ml) . The secreted fuεion protein was rapidly purified by a single IgG affinity purification step. The engineering of a collagenase cleavage recognition consenεus sequence between these two polypeptides facilitated the cleavage of the fusion protein so that the purified human α-Gal A polypeptide could be readily separated from the protein A domain by a second IgG purification step. Of interest was the fact that the fusion construct retained α-Gal activity, presumably indicating that the enzyme polypeptide formed the active homodimeric configuration even though the carboxy terminus was joined to an additional 56 residues of the protein A domain. Since COS-l cells transfected with an α-Gal A construct exhibit similar levels of expresεion and diεtribution between cellε and media it appears that the protein A domain does not interfere with either the folding or the proper processing of this lysosomal enzyme. Furthermore, the presence of the dimerized α- Gal A polypeptide did not inhibit the binding of the protein A domain to the IgG affinity column. The insertion of the four residue collagenase cleavage recognition sequence between the α-Gal A and protein A polypeptides permited cleavage of the fusion protein leaving only two of the collagen residues on each of the peptides. The ease of cDNA construction using the polymerase chain reaction, transfection and purification of the expressed protein permits the isolation of small, but sufficient amount of α-Gal A for characterization of the enzyme's physical and kinetic properties. Using site-directed mutagenesis or naturally occuring mutant εequences, this system provides a reasonable approach to determine the effects of the altered primary structure on the function of the protein. Fusion constructs with the protein A domain E preceeding the amino terminus and the following the carboxy terminuε may also be engineered to evaluate which fusion construct will interfere the least, if at all, with the protein'ε biologic function and the ability to bind IgG. Uεing thiε aεpect of the invention, any cleavage εite or enzyme cleavage εubstrate may be engineered between the α-Gal A sequence and a second peptide or protein that has a binding partner which could be used for purification, e.g.. any antigen for which an immunoaffinity column can be prepared. 5.2.4. CHARACTERIZATION OF THE RECOMBINANT ENZYME The purified recombinant enzyme produced in the mammalian expression systems described herein (e.g.. the CHO expreεεion system) , had molecular weight, pH optimum, km and isoelectric point values which were esεentially identical to those of the enzyme purified from the human plasma (Bishop, et al., 1978, Biochim. Biophys. Acta. 525: 399; Bishop and Desnick, 1981, J. Biol. Chem. 256:1307). Analyεis of the carbohydrate moieties on thiε enzyme revealed the preεence of three oligoεaccharide chainε on the α-Gal A polypeptide. Theεe chainε were a mixture of complex, hybrid and high-mannose types aε evidenced by endoglycosidase and QAE Sephadex studies. Most importantly, the recombinant enzyme was also similar to the native plasma form of α-Gal A in having terminal sialic acid moieties (Bishop & Desnick, 1981, J Biol. Chem. 256: 1307) . In the limited clinical trial described supra. the plasma form of the enzyme was shown to be more effective in degrading circulating GbOse 3 Cer than the splenic form. Therefore, the recombinant enzyme or a modified recombinant enzyme, including but not limited to modifications of its carbohydrate chains or amino acid sequence, may be the most appropriate form for enzyme replacement therapy of Fabry disease. Indeed, the saturable uptake of recombinant α-Gal A by Fabry and normal fibroblastε is demonstrated in the examples herein, and is εhown to be εpecifically inhibited by 2 mM mannoεe-6-phoεphate. In addition, the CHO expreεεion εyεtem deεcribed herein has great promise for studieε of the cell biology of lyεoεomal biogeneεiε and glycohydrolase processing. Light microscopy revealed highly vacuolated cytoplasm in the DG5.3 CHO cells suggesting a proliferation of lysoεomal membraneε and offering the potential for analyεiε of lyεoεomal biogenesis. Preliminary studieε have indicated that the recombinant enzyme iε εyntheεized very rapidly, exitε the endoplasmic reticulum in 5-10 min following its syntheεiε and iε secreted 45-60 min later. These fast kinetics of recombinant α-Gal A biosynthesis allow for interesting studieε involving lysosomal enzyme biosyntheεiε and offer a methodology that, to date, iε only rivaled by viral systems. In fact, recombinant α-Gal A is synthesized so rapidly that a single radioactive pulse of 3 min iε εufficient to label enough enzyme for theεe εtudies. The unexpectedly specific secretion of only the overproduced recombinant α-Gal A and not other lysoεomal enzymeε appearε analogouε to "gene dosage-dependant secretion" described by Rothman, et al. (Stevenε et al., 1986, J. Cell Biol. 102:1551; Rothman et al. , 1986, Proc. Natl. Acad. Sci. USA 83:3248) and poεes interesting questionε which can be evaluated in thiε system. 5.2.5. MODIFIED GLYCOFORMS OF RECOMBINANT α-Gal A FOR ENZYME THERAPY IN FABRY DISEASE Initial experiments to assess the clearance kinetics and tissue distribution of recombinant α-Gal A in mice revealed 50% targeting to the liver with the remaining enzyme being distributed to many other tissues including significant targeting to kidney, heart and skin. While this distribution is similar to that previously observed for the plasma form of human α-Gal A in mice, it may be appropriate to modify the enzyme for altered tisεue targeting. Modificationε of the recombinant α-Gal A to enhance tiεεue targeting including εelective deglycosylation of the complex and high mannose carbohydrate moieties covalently attached to the recombinant enzyme. In particular, the invention includes modification of host cells that allow for expresεion of εialylated and asialylated glycoforms of the enzyme, both of which may be easily purified (see Section 9 infra) . For example, when using CHO cells to express α-Gal A, the CHO cells may be co-transfected with εialyl-transferase gene construct that supplies the misεing function to the CHO cell in order to express the sialylated glycoform of α-Gal A. Alternatively, sequential deglycosylation to various glycoforms for use in the treatment of Fabry diseaεe. Such modifications have proven to be important in effectively targeting β- glucocerebroεidaεe to acrophageε in the treatment of Gaucher disease (Barton, N.W. , et al. , 1990, Proc. Natl. Acad. Sci. USA 87: 1913). In this case, placenta derived 3-glucocerebrosidaεe was sequentially treated with neuraminidase, 3-galactosidase and N-S- acetylglucosaminidaεe to expose terminal mannose residueε for uptake by the mannose receptor of these cells (Stahl, et al. , in The Molecular Baεiε of Lyεoεomal Diεorders, Barranger, J.A. and Brady, R.O. eds., 1984 Academic Press, NY pp. 209-218). Modifications to human recombinant α-Gal A included in the scope of this invention include, but are not limited to, sequential deglycosylation by neuraminidase to expose terminal galactose; β- galactosidaεe treatment to expose N-/3- acetylglucoεaminyl reεidues; and N-/3- acetylglucoεaminidaεe treatment to expose mannose residues for specific targeting and uptake by various cell types. The sequentially deglycosylated recombinant α-Gal A glycoforms may be analyzed by determining the clearance kinetics and tissue distribution of each of the radiolabeled glycoforms following intravenous administration in mice and monkeys. Deglycosylation of recombinant α-Gal A may be accomplished in a number of wayε. The general methods of sequential treatment by exo-glycosidaεeε which may be uεed are eεsentially those previouεly deεcribed (Murray, G. J. , 1987, Meth. Enzymol, 149: 25). For example, terminal sialic acid residues can be removed by treatment with neuraminidase covalently bound to agarose; e.g. , type VI neuraminidaεe attached to agarose (SIGMA Chemical Co. , St. Louis, MO) may be uεed at 40 U/g to treat 100 mg α-Gal A with 8 unitε of conjugated neuraminidaεe at pH 5.0 for 4 hour at 37"C. The conjugated neuraminidaεe can be removed by centrifugation. Similarly, /3-galactoεidase (3 Units per 100 mg α-Gal A) purified from Streptococcus pneumoniae may be used to remove terminal galactose residueε. Finally, jack bean N-/5- acetylglucosaminidase (SIGMA Chemical Co. , St. Louiε, MO) can be uεed; e.g.. 3 X 10 6 unitε can be mixed with each 100 mg aliquot of the recombinant α-Gal A for four hours at 37~C. At each step, the recombinant enzyme can be rapidly purified free of deglycosylating enzymes and free carbohydrate by purification over the α-galactosylamine-Sepharoεe affinity column. For the analysis of the in vivo fate of the various glycoforms, including plasma clearance kinetics and tisεue diεtribution studies, the recombinant α-Gal A may be labeled prior to modification. For example, the recombinant α-Gal A can be radiolabelled by growth in the CHO DG5.3 cell 0 line in the presence of 50 μCi/ml [ 3S S]methionine (>1000 Ci/mmole) for 24 hours. The secreted radiolabeled enzyme can be purified from the harvested media by α-galactosylamine-Sepharose affinity chromatography as previously described. Essentially 5 100% of the radiolabelled protein secreted by these cells is α-Gal A which can then be used for the sequential generation of the glycoforms. 5.3. USES OF THE RECOMBINANT α-Gal A o The purified products obtained in accordance with the invention may be advantageouεly utilized for enzyme replacement therapy in patientε with the lyεoεomal εtorage diεorder, Fabry Disease. Alternatively, the purified products obtained in 5 accordance with the invention may be used in vitro to modify α-D—galacto-glyconjugates in a variety of processes; e.g.. to convert blood group B erythrocytes to blood group O; in commerical processeε requiring the converion of sugars such as raffinose to sucrose 0 or melibiose to galactoεe and glucose; etc. These are diεcuεεed in more detail in the subsectionε below. 5.3.1. α-Gal A ENZYME THERAPY IN FABRY DISEASE Among the inborn errorε of metabolism, studieε of 5 patientε with lyεosomal storage disorderε have provided baεic underεtanding of the biology of the lysosomal apparatus and its hydrolases, their biosynthesiε and processing (Rosenfeld, et al., 1982, J. Cell Biol. 93: 135; Lemansky, et al., 1984, J. Biol. Chem. 259: 10129), the mechanisms of their transport to the lysosomes (Neufeld, et al., 1975, Ann. Rev. Biochem. 44: 357; Sly et al. , 1982, J. Cell Biochem. 18:67; Kornfeld, S. , 1986, J. Clin. Invest. 77: 1), and their cofactor requirements (Verheijen, et al., 1985. Eur. J. Biochem. 149: 315; d'Azzo, et al., 1982, Eur. J. Biochem. 149: 315; Mehl, et al., 1964, Physiol. Chem. 339: 260; Conzelman, et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3979). Of the over 30 lyεoεomal εtorage diεorderε, Fabry diεeaεe iε an ideal candidate for the application of the recombinant DNA techniqueε described herein to evaluate and utilize various therapeutic approaches in model systems, aε well as to correlate the effects of site-specific changes on enzyme structure and function. The disease nas n ° central nervous system involvement; thus, the blood/brain barrier does not present an obstacle to enzyme replacement therapy. The defective enzyme, α- Gal A, is a homodimer (Bishop & Deεnick, 1981, J. Biol. Chem. 256: 1307) , in contrast to some lysosomal enzymes which have different subunits such as β- hexoεaminidaεe A (Mahuran, et al., 1982, Proc. Natl. Acad. Sci. USA 79: 1602); therefore, only a single gene product must be obtained. The metabolic defect in cultured fibroblastε from Fabry diεease has been corrected in vitro by the addition of exogenous enzyme into the.culture medium (Cline, et al., 1986, DNA 5: 37) . Alεo, atypical variantε with Fabry diεeaεe have been identified, theεe maleε are clinically aεymptomatic, having εufficient reεidual α-Gal A activity (3 to 10%) to protect them from the major morbid manifeεtations of the disease (Lemansky, et al., 1987, J. Biol. Chem. 262:2062; Clarke, et al., 1971, N. Engl. J. Med. 284: 233; Romeo, et al., 1975, Biochem. Genet. 13: 615; Biεhop, et al., 1981, Am. J. Hum. Genet. 71: 217A; Bach, et al., 1982, Clin. Genet. 21: 59; and, Kobayaεhi, et al., 1985, J. Neurol. Sci. 67: 179). Finally, aε noted above, limited human trialε have demonstrated the biochemical effectivenesε of enzyme replacement to deplete the circulating εubεtrate prior to vascular deposition as well as the absence of immunologic complicationε (Brady, et al., 1973, N. Engl. J. Med. 289: 9; Desnick, et al., 1979, Proc. Natl. Acad. Sci. USA 76:5326; Bishop, et al., 1981, Enzyme Therapy XX: In: Lysoεomeε and Lysosomal Storage Diseases, Callahan, J.W. and Lowden, J.A. , (eds.). Raven Presε, New York, pp. 381; Deεnick, et al., 1980, Enzyme Therapy XVII: In: Enzyme Therapy in Genetic Diεease: 2, Desnick, R.J. (ed.), Alan, R. Liss, Inc., New York, pp. 393). I these studieε, both splenic and plasma isoforms of the α-Gal A enzyme were adminiεtered intravenouεly. The circulating half-life of the εplenic isozyme was about 10 min whereas that for the plasma isozyme was approximately 70 min. After each dose of the splenic isozyme, the concentration of the accumulated circulating subεtrate decreaεed maximally in 15 min. In contraεt, injection of the plasma isozyme decreased circulating substrate levels gradually over 36 - 72 hours. Since the secreted form of the recombinant α-Gal A appears to be similar to the plasma isozyme, the secreted form of the recombinant enzyme could be effective for the long term depletion and control of circulating subεtrate levels. The dose of the partially purified plasma and splenic isozymes administered in the above clinical trials was 2000 U/kg body weight, or a dose equivalent to giving 1 μg/kg of pure enzyme. Since this dose proved effective in reducing the level of circulating subεtrate, a εimilar doεe of the recombinant enzyme εhould have a εimilar effect. However, the recombinant enzyme could be adminiεtered at a dosage ranging from 0.1 μg/kg to about 10 mg/kg and, preferably from about 0.1 mg/kg to about 2 mg/kg. The ability to produce large amounts of the recombinant enzyme in accordance with this invention will permit the evaluation of the therapeutic effect of significantly larger doseε. 5.3.2. IN VITRO USES OF α-Gal A α-Gal A iε a galactoεyl hydrolaεe which haε activity toward variouε oligoεaccharideε, glycoproteinε, glycopeptides and glycolipids with terminal α-galactosidic linkageε. Thuε, the enzyme can be used in vitro to modify these α-galacto- glycoconjugateε. For example, the recombinant α-Gal A of the invention could be utilized for a variety of deεirable modifications including but not limited to: (a) the conversion of blood group B erythrocytes to cells expresεing the blood group O antigen (Harpaz, et al., 1977, Eur. J. Biochem. 77:419-426); and (b) the hydrolyεiε of stacchyose to raffinoεe, raffinoεe to the diεaccharide εucroεe, or the hydrolyεiε of melibiose to galactose and glucose (Silman, et al., 1980, Biotechnol. Bioeng. 22:533). Such hydrolyses have commercial applications as in the degradation of molasεeε aε a substrate for yeast production (Liljeεtrom-Suominen, et al., 1988, Appl. Environ. Micro. 54:245-249). 6. EXAMPLE: OVEREXPRESSION AND SPECIFIC SECRETION OF BIOLOGICALLY ACTIVE α-GALACTOSIDASE A IN A MAMMALIAN CELL SYSTEM The subεectionε below deεcribe the production of large quantities of human recombinant α-Gal A. A full-length cDNA encoding human α-Gal A waε inserted into the expression vector p91023(B) in front of the amplifiable dihydrofolate reductase (DHFR) cDNA. The functional integrity of cDNA conεtruct (p91-AGA) was confirmed by transient expression of active enzyme in COS-l cells; 650 U/mg (nmol/hour) versus endogenous levels of -150 U/mg of 4-MU-α-D-galactopyranoside activity. The p91-AGA construct waε introduced by electroporation into DG44 dhfr" CHO cellε. Positive selection in media lacking nucleosideε resulted in the isolation of clones expresεing the active enzyme at levelε ranging from 300 to 2,000 U/mg. Selected subclones, grown in increasing concentrations of methotrexate (MTX, 0.02 to 1.3 μM) to co-amplify DHFR and α-Gal A cDNAs, expressed intracellular levels of α-Gal A activity ranging from 5,000 to 25,000 U/mg. Notably, subclone DG44.5, which expresεed high intracellular levels of α-Gal A, secreted more than 80% of the total recombinant enzyme produced. At a MTX concentration of 500 μM, 10 7 cellε εecreted -15,000 U/ml culture media/day. Of note, endogenouε CHO lysosomal enzymes were not secreted including β- hexosaminidase, α-mannosidase, 3-galactoεidaεe and β- glucuronidaεe, indicating that the εecretion was α-Gal A specific and not due to saturation of the mannose-6- phoεphate receptor-mediated pathway. Uεing a hollow fiber bioreactor, up to 5 mg per liter per day of recombinant α-Gal A enzyme waε produced per day. The εecreted α-Gal A was purified by affinity chromatography for characterization of various physical and kinetic properties. The recombinant α- Gal A had a pi, electrophoretic mobility and Km values which were similar to the enzyme purified from human plasma. In addition, 32 P labeling studies revealed that both the lysoεomal and εecreted forms were phosphorylated, preεumably in their oligoεaccharide moieties. Current studieε are directed to characterize additional kinetic and physical properties, the oligosaccharide moieties and the crystal structure of the recombinant enzyme. Furthermore, the availability of large amounts of soluble active enzyme will permit the evaluation of enzyme replacement in animal systems prior to clinical trials in hemizygotes with Fabry diεeaεe. 6.1. MATERIALS AND METHODS 6.1.1. MATERIALS Restriction endonucleaseε, the Klenow fragment of DNA polymeraεe I, T4 polymeraεe and T4 ligaεe were from New England Biolabε; α and 7-32[P] dNTPε (3000 Ci/mole) and α35[S]dATP (100 Ci/mole) were from Amerεham. The COS-l cell line was purchased from ATCC, Rockville, MD. The CHO DG44 dhfr ' cell line iε deεcribed (Urlaug, et al., 1986, Somat. Cell Genet. 12:555-566) . 6.1.2. CONSTRUCTION OF EXPRESSION VECTOR P91-AGA Plasmid pcDAG126 (Bishop, et al. , 1988, in, Lipid Storage Disorders, Salvaryre, R. , Douste-Blazy, L. Gatt, S. Eds. Plenum Publiεhing Corporation, New York, pp. 809 to 822) containing the full-length α-Gal A cDNA was digested with Bam HI and Pst I and the 1.45 kb insert fragment was purified by agarose gel elctrophoresiε. The cDNA was then force-subcloned into plasmid pGEM-4 at the Bam HI and Pst I sites reεulting in pGEM-AGA126. Thiε plaεmid was then digested with Hind III, end-filled using Klenow and ligated to Eco RI linkers. After digestion with Eco RI, the 1.45 kb fragment was purified as above and cloned into the Eco RI site of the mammalian expression vector p91023(B) (Wong et al., 1985, Science 228:810) resulting in p91-AGA (FIG. 1G) . 6.1.3. CELL CULTURE, ELECTROTRANSFECTION, AND GENE AMPLICATION COS-l and DG44 CHO cells were maintained at 37°C in 5% C0 2 in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum (FCS) and antibiotics; DG44 (dhfr " ) cells were maintained by addition of 0.05 mM hypoxanthine and 0.008 mM thymidine to the media. Following transfection, the recombinant CHO lines were grown in DMEM supplemented with 10% dialyzed FCS in the absence or presence of MTX. For electroporation, cells were trypsinized and centrifuged at 800 x g at room temperature for 10 min. The pellet was washed once with DMEM εupplemented with 10% FCS serum and twice in ice-cold electroporation buffer (phosphate buffered sucrose; 272 mM sucrose, 7 mM εodium phosphate, pH 7.4, containing 1 mM MgCl 2 ) . Cells were then resuspended in phoεphate buffered εucrose at -0.65 to 1.0 x 10 7 /ml. The cell suεpenεion (0.8 ml) waε placed in a 0.4 cm gap cuvette (Bio-Rad) , 5-20 μg of plaεmid DNA waε added and kept on ice for 10 min. The cuvette was placed in the "Gene Pulser" chamber (Bio-Rad) and pulsed once at 25 μF with 300 V for COS-l cells or 400 V for CHO DG44 (dhfr " ) cells, the optimized settings for the respective cell lines. The cuvette containing the pulsed cells was placed on ice for 10 min and then the cells were removed from the cuvette and placed in 15 ml of DMEM supplemented with 10% FCS. For transient expression, COS-l cells were harvested at 72 hours and assayed immediately. For stable expression, the transfected DG44 cells were grown for 48 hours and then were removed from the culture dish by trypsinization and replated at a 1:15 ratio in DMEM εupplemented with 10% dialyzed FCS. 0 Media waε replaced every four days. After two weeks of growth, cell foci became visible and individual cloneε were isolated with cloning rings. Clones which expressed the highest levels of α-Gal A were subjected to amplification en masse by step-wise growth in 5 increasing concentrations of methotrexate (MTX), 0.02, 0.08, 1.3, 20, 40, 80, 250 and 500 μM. 6.1.4. ENZYME AND PROTEIN ASSAYS For enzyme assay, the cells in a 100 mm culture o dish were washed twice with 5 ml of phosphate buffer saline (PBS) and scraped into a 12 ml conical tube using a rubber policeman. Following centrifugation at 800 x g for 10 min, the cells were resuspended in 1 ml of 25 mM NaP0 4 buffer, pH 6.0, and then disrupted in a 5 Branson cup sonicator with three 15 second burstε at 70% output power. The sonicate was centrifuged at 10,000 x g for 15 min at 4°C and the supernatant was removed and assayed immediately. Alternatively, for rapid screening, cells were washed as above and 1 ml 0 of lysiε buffer (50 mM εodium phoεphate buffer, pH 6.5, containing 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 0.2 mM PMSF) was added to the diεh. The lyεed cellε were incubated at 4°C for 30 min, the lyεates collected and transfered to a 1.5 ml tube, centrifuged 5 in a microfuge, and then the εupernatant was removed for aεsay. The α-Gal A activities in the cell lyεateε and media were determined uεing 5 mM 4-methylumbelli- feryl-α-D—galactopyranoεide (4MU-α-Gal) aε previously described (Bishop, et al., 1980, In Enzyme Therapy in Genetic Diseases: 2. Desnick, R.J. (Ed.) . Alan R. Liss, Inc. New York, p. 17) . Briefly, a εtock solution of 5 mM 4MU-α-Gal was prepared in 0.1M citrate/0.2M phosphate buffer, pH 4.6, in an ultraεonic bath. The reaction mixture, containing 10 to 50 μl of cell extract and 150 μl of the εtock εubεtrate εolution, waε incubated at 37°C for 10 to 30 min. The reaction was terminated with the addition of 2.3 ml of 0.1 M ethylenediamine. The fluorescence was determined using a Turner model 111 Fluorometer. One unit of activity is the amount of enzyme which hydrolyzes one nmol of substrate per hour. The activities of α-mannosidase, β-galactosidase, β- hexosaminidase, jδ-glucuronidase and acid phosphataεe were determined uεing the appropriate 4- methylumbelliferyl substrate. Protein concentrations were determined by the fluorescamine method (Bohlen, et al., 1973, Arch. Biochem. Biophys. 155: 213) as modified by Bishop et al. (Bishop, et al., 1978, Biochim. Biophys. Acta 524: 109) . 6.2. RESULTS 6.2.1. EXPRESSION OF HUMAN α-Gal A IN COS-l CELLS The full-length human α-Gal A cDNA was cloned into the expresεion vector p91023(B) (Wong, et al., 1985, Science 228: 810) and the conεtruct, designated p91-AGA, was introduced into COS-l cells by electroporation. Increased levels of α-Gal A activity were detected at 24, 48 and 72 hours after tranεfection (FIG. 2A) , indicating the functional integrity of the p91-AGA construct. At 72 hours after transfection, the α-Gal A activity increased about four-fold, while no increase in α-Gal A activity was observed in cells transfected with the p91023(B) vector containing the α-Gal A cDNA in the antisense orientation, nor in the cells that received no DNA. In addition, the jS-galactosidaεe levels, determined as a lysosomal enzyme control, were not changed (FIG. 2B ) • 6.2.2. TRANSFECTION AND AMPLIFICATION OF α-Gal A IN DHFR " CHO CELLS Recombinant clones stably expresεing human α-Gal A were obtained by electrotransfection of the p91-AGA construct into DG44 dhfr " CHO cells and amplification of the integrated vector DNA with selection in increasing MTX concentrations. Initial growth in media lacking nucleosides resulted in the identification of over 100 clones expreεεing α-Gal A at levelε ranging from 100 to 1,800 U/mg protein (Table I) . Clones with the highest α-Gal A level were grown in the presence of 0.02 to 0.08 μM MTX to amplify the integrated p91-AGA DNA. Table II shows that the intracellular α-Gal A levels in repreεentative amplified cloneε increased 2 to 6 fold in 0.02 μM MTX and up to 10 fold when further amplified in 0.08 μM MTX. TABLE I Intracellular α-Galactoεidaεe A Activity In DG 44 (dhfr " ) CHO Cellε* Following Elecrotranεfection with p91-AGA CLONE α-Gal A Activity (U/mg protein) Parental DG44: 497 Transfected: 4 493 5 1,243 7 108 8 524 9 1,155 11 1,115 20 624 24 1,864 46 720 52 180 * Cells grown in DMEM supplemented with 10" dialyzed FCS. TABLE II Intracellular α-Galactosidase' A Activities In p91-AGA Tranεfected DG44 (dhfr) CHO Cells Following Initial Applification In Methotrexate CLONE α-Gal A (U/mg) 0.02 UM MTX: 5 4,990 9 2,900 11 3,170 46.1 1,230 46.5 4,570 46.12 4,100 0.08 uM MTX: 5.3 23,400 5.7 7,950 5.9 14,680 5.11 3,070 9.1 10.290 9.4 7,950 9.6 3,860 6.2.3. HIGH LEVEL EXPRESSION CLONES SECRETE HUMAN α-Gal A Among the poεitive cloneε amplified in the presence of 0.08 μM MTX, clone 5.3 had the highest intracellular α-Gal A level (Table II) and therefore was chosen for further amplification. When grown in the presence of 1.3 μM MTX, the α-Gal A activity in the growth media of clone DG5.3 was determined to be 2,500 U/ml, or 25-fold greater than the level in untransfected parental DG44 cells (50 to 100 U/ml) . Growth in the presence of increasing concentrations of MTX, resulted in increased intracellular and secreted α-Gal A activities (Table III) . Interestingly, over 80% of the total α-Gal A produced was secreted and growth in increasing MTX concentrations continued to increase the percentage of enzyme secreted. Note that the data shown in Table III were obtained after the cells were amplified in the presence of the indicated MTX concentration and then asεayed for α-Gal A activity after growth for three weeks in the absence of MTX, which accounts for their lower intracellular activites than during growth under εelective pressure (Pallavicini, et al., 1990, Mol. Cell Biol. 10: 401; Kaufman, R.J. , 1990, Meth. Enzymol, 185: 537; Kaufman, R.J-/ 1990, Meth. Enzymol, 185: 487). TABLE III Intracellular And Secreted α-Galactosidase A Activities In p91-AGA Transfected CHO Line DG5.3 Following Step-Wise Amplification In Methotrexate. Data Were Obtained On Clones After Three Weeks Of Growth In The Absence Of Methotrexate. Methotrexate Concentration CHO Cellε Media* (μM) (U/mg) (U/ml) Untranεfected DG44: 250 100 10 7 cellε and 10 ml of media for each Methotrexate concentration. 6.2.4. SPECIFIC SECRETION OF OVER- EXPRESSED LYSOSOMAL ENZYMES To determine whether the secretion of α-Gal A was due to saturation of the receptors for lysosomal targeting, the culture media from clone DG5.3 was assayed for the presence of other lysosomal enzymes. As shown in Table IV, the activities of seven representative lysosomal enzymes were not increased or were lower than those in the media of the DG44 parental cell line, indicating that the DG5.3 secretion of α-Gal A was specific. To determine if the εecretion was specific to clone DG5.3, another clone, DG9, which was not secreting α-Gal A (i.e., activity in media was 120 U/ml) , was subjected to step-wiεe growth in increaεing MTX concentrations (i.e., from 0.02 to 20 μM MTX). After amplification in 20 μM MTX, clone DG9 had intracellular and secreted levels of α-Gal A ativity of 9,400 U/mg and 7,900 U/ml, respectively; i.e. 89% of the total α-Gal A activity produced was secreted. TABLE IV Lysosomal Enzyme Activities Secreted In Culture Media Of Transfected CHO Cellε * Average of Triplicate Determinations in Two Independent Experiments. Since treatment of recombinant CHO cells with 50 mM butyrate has been shown to specifically increase tranεcription of the stably integrated p91023(B) s vector in CHO cellε (Dorner, et al., 1989, J. Biol. Chem. 264: 20602; Andrews & Adamson, 1987, Nucl. Acids Res. 15: 5461) another transfected clone, DG11, which was not amplified, was grown in the presence of 5 mM butyrate (Table V) . The intracellular levels of α-Gal 0 A activity increased from 259 U/mg to 687 U/mg. Notably, in the presence of butyrate, increased α-Gal A activity was secreted into the media (103 to 675 U/ml) , suggesting that secretion occured when the gene copy number increased (or, more preciεely, the steady 5 state of α-Gal A mRNA was increased) . Incubation of butyrate-induced cells with 5 mM M-6-P (to prevent recapture of the secreted enzyme by the cell surface receptor) did not result a significant increase in the amount of α-Gal A secreted. 0 TABLE V Butyrate Effect On α-Gal A Secretion in CHO DG11 α-Gal A Activity 5 Clone Cells Media Control o Butyrate Butyrate + 5 mM M-6-P 6.2.5. EFFECT OF SERUM CONCENTRATION ON SECRETION 5 To determine if the serum concentration of the growth media had an effect on the levels of recombinant α-Gal A secretion, clone DG5.3 was grown in 100 mm culture disheε at a density of 5 x 10 6 cellε per diεh, in the preεence of 0% to 10% dialyzed FCS for 5 dayε. There was no apparent effect on α-Gal A secretion in cells grown with 2.5% to 10% serum (FIG. 3A-3B) . The decreased level of secretion by DG5.3 cells cultured in 0% and 1% serum presumably reflected the poor growth of these cells. 6.2.6. PRODUCTION IN BIOREACTORS To produce large quantitieε of recombinant human α-Gal A, 10 8 cells of clone DG5.3 which had been grown in the presence of 500 μM MTX (DG5.3 500 ), were used to seed a hollow fiber bioreactor. As shown in FIG. 4, the level of α-Gal A produced increased to about 10,000 U/ml per day. This level remained constant for about three months. In addition, the serum concentration required by these cells in the bioreactor was step-wise decreased to 1% without seriously decreasing α-Gal A production (FIG. 4) . A single 90-day run of this bioreactor reεulted in >350 mg of active recombinant α-Gal A secreted into the culture media. 6.3. DISCUSSION For human α-Gal A, post-translational modificationε appear to be essential for stability and activity, as evidence by the fact that the unglycosylated enzyme expressed in E^ . coli was unstable and rapidly degraded (Hantzopoulos & Calhoun, 1987, Gene 57: 159) . In addition, the α-Gal A subunit, which has four potential N-glycosylation siteε, undergoes carbohydrate modification and phoεphorylation for lyεoεomal delivery (Lemanεky, et al., 1987, J. Biol. Chem. 262: 2062) . Previouε characterization of α-Gal A purified from plasma and tissue identified their different carbohydrate compositions, the plasma glycoform having more sialic acid residues (Bishop, et al., 1978, Biochim. Biophys. Acta 524: 109; Bishop, et al., 1980, Birth Defects 16:1; p. 17; Bishop and Desnick, 1981, J. Biol. Chem. 256:1307). Moreover, clinical trials of enzyme therapy revealed that compared to the tissue-derived form, the plasma glycoform had a prolonged retention in the circulation and was more effective in depleting the circulating accumulated subεtrate following intravenouε adminiεtration to patentε with Fabry diεease (Desnick, et al., 1979, Proc. Natl. Acad. Sci. USA 76: 5326) . Thus, the amplified expresεion of human α-Gal A in CHO cellε waε choεen for the expreεεion of thiε recombinant enzyme whoεe native compoεition includes galactosyl and sialic acid residues (Ledonne, et al. , 1983, Arch. Biochem. Biophys. 224:186). Although this is the first human lysoεomal hydrolase to be succeεεfully overexpreεεed, an unexpected finding was the secretion of over 80% of the enzyme produced. This could result from several different mechanisms including (a) saturation of the mannoεe-6-phosphate receptor pathways; (b) a mutation that alters a critical glycosylation site; (c) failure to expose the mannose-6-phoεphate moiety for receptor binding; or (d) an unuεually low affinity of recombinant α-Gal A for the mannoεe-6-phosphate receptor (Reitman & Kornfeld, 1981, J. Biol Chem. 256: 11977; Lang, et al., 1984, J. Biol. Chem. 259: 14663; and, Gueze, et al., 1985, J. Cell. Biol. 101: 2253; for review see, Kornfeld & Mellman, 1989, Ann. Rev. Cell. Biol. 5: 483). If the secretion of α-Gal A was due to the saturation of the receptor-mediated pathway, then it would be expected that the other endogenous lysosomal enzymes also would be secreted. However, the levelε of secreted CHO hydrolases were unchanged, or decreaεed (Table IV) . To rule out a possibible mutation in the α-Gal A cDNA introduced during conεtruction and integration of the vector (Caloε, et al., 1983, Proc. Natl. Acad. Sci. USA 80: 3015) , the integrated vector DNA waε amplified by the polymerase chain reaction. Ten subclones were completely sequenced in both orientations, and no mutations were identified. In companion studies of the purified recombinant protein (described infra) , it was shown that the mannose-6-phosphate moiety was present on the enzyme and that the enzyme bound efficiently to the immobilized mannose-6-phosphate receptor. Furthermore, to prove that the secretion of this protein in the expression system utilized was not α-Gal A dependent, the cDNA encoding another lysosomal hydrolase α-N-acetylgalactosarainidase, was inserted into p91023(B) and amplified in CHO cells. Analogous to the observations with α-Gal A, cells that were high expresεorε of α-N-acetylgalactoεaminidaεe (α-GalNAc) , alεo secreted the recombinant enzyme in the medium. The presence of functional mannose-6-phoεphate moietieε on the secreted enzyme implied that perhaps a different mechanism was responsible for its secretion. In fact, many other secreted proteins have been shown to contain mannose-6-phosphate. Some of these proteins include lysoεomal proteins while the location of others is not clear. These proteins include, proliferin (Lee & Nathans, 1988, J. Biol. Chem. 263: 3521) secreted by proliferating mouse placental cell lineε; epidermal growth factor receptor in A-431 cellε (Todderud & Carpenter, 1988, J. Biol. Chem. 263: 17893) ; tranεforming growth factor βl (Purchio, et al. , 1988, J. Biol. Chem. 263: 14211) ; uteroferrin, an iron containing acid phosphatase secreted in large amounts by the uterine endometrium of pigs (Baumbach, et al., 1984, Proc. Natl. Acad. Sci. USA 81: 2985); and cathepsin L (MEP) , a mouεe lysosomal cystein protease secreted by mouse NIH 3T3 fibroblastε (Sahagian & Gottesman, 1982, J. Biol. Chem. 257: 11145) . Of interest, transformation of NIH 3T3 cellε with Kirεtein virus resultε in a 25-fold increaεe in the synthesis of MEP causing thiε enzyme to be εelectively εecreted even though it containε functional mannoεe-6-phosphate moieties (Sahagian & Gottesman, 1982, J. Biol. Chem. 257: 11145). Recently, the mechanism for the selective secretion of MEP has been identified and it involves an inherent low affinity of MEP for the mannose-6-phosphate receptor (Dong, et al., 1989, J. Biol. Chem. 264: 7377) . It is also notable that the plasma-directed overexpresεion of yeaεt vacuolar carboxypeptidase Y in yeaεt reεults in over 50% of the normally glycoεylated protein secreted as the precursor form (Stevens, et al., 1986, J. Cell. Biol. 102: 1551). Similar findings were observed for the yeast proteinase A gene (Rothman, et al., 1986, Proc. Natl. Acad. Sci. USA 83: 3248) . Studieε have εuggeεted that the precursor glycoproteins have εubcellular localization εignalε located within the N-terminal propeptide that are recognized by the εecretion pathway, thereby precluding delivery to the lysosome-like vacuole. It iε notabie that the εecretion of theεe yeaεt genes iε gene-dosage dependent and that a similar phenomenon is observed for the expresεion in CHO cells of human α- Gal A. Also, it is of intereεt that the precurεor form of the yeast enzymes was secreted. The plasma form of α-Gal A is more sialyated and secreted, and others have shown that the lysosomal enzymes in human urine are the precursor forms (Oude-Elferink, et al., 1984, Eur. J. Biochem. 139: 489). However, N-terminal sequencing of recombinant α-Gal A expresεed by DG44.5 revealed that the amino-terminus was identical to that of α-Gal A purified from human lung (Bishop, et al. , 1986, Proc. Natl. Acad. USA 83:4859). Thus, it is posεible that the high-level expreεεion of human lyεoεomal hydrolaεeε reεultε in their εecretion due to the inability to modify the precurεor and/or inability of the εubcellular localization machinery to accommodate the intracellular delivery of the overexpressed glycoprotein. However, this again would result in the secretion of other lysosomal enzymes. Since no other lyεoεomal enzymeε are detected in the culture media, it iε leεε likely that εecretion of α- Gal A results from saturation of a component of the subcellular localization machinery. Further studieε, directed to determine amino acid, carbohydrate or other differences (e.g. , sulfation) between the secreted and intracellular forms of recombinant α-Gal A may provide insights into the mechanism underlying the mislocalization and εelective secretion of human α-Gal A. In addition, efforts to evaluate the generality of this observation should include the overexpression of other human lysosomal enzymes. The fact that large amounts of recombinant human α-Gal A are secreted by CHO cells permits the convenient production of the recombinant enzyme. Section 8, infra, describeε a method for the purification of the recombinant enzyme and the characterization of itε phyεical and kinetic propertieε including itε receptor-mediated uptake by Fabry fibroblaεtε. 7. EXAMPLE: PURIFICATION, CHARACTERIZATION AND PROCESSING OF RECOMBINANT α-GalACTOSIDASE A The εubεections below describe the purification of human α-galactosidase A cloned into the amplifiable eukaryotic expression vector, p91023(B), and overexpressed in Chinese hamster ovary (CHO) cells. The recombinant enzyme protein, was selectively secreted into the culture media and over 200 mg waε purified to homogeneity by a Fast Protein Liquid Chromatographic procedure including affinity chromatography on α-galactosylamine-Sepharose. The purified secreted enzyme was a homodimeric glycoprotein with native and subunit molecular weights of about 110 and 57 kDa, respectively. The recombinant enzyme had a pi of 3.7, a pH optimum of 4.6, and a km of 1.9 mM toward 4-methylumbelliferyl-α- D—galactopyranoside. It rapidly hydrolyzed pyrene- dodecanoyl-sphingosyl-trihexoside, a fluorescently labeled analogue of the natural glycosphingolipid εubstrate, which waε targeted with apolipoprotein E to the lyεosomes of the enzyme-producing CHO cells. Pulse-chase studies indicated that the recombinant enzyme assumed its disulfide-defined secondary structure in <3 min, was in the Golgi by 5 min where it became Endo H resiεtant and waε secreted into the media by 45-60 min. Both the intracellular and secreted forms were phosphorylated. The secreted enzyme subunit was slightly larger than the intracellular subunit. However, following endoglycosidase treatment, both subunitε co-migrated on SDS-PAGE, indicating differenceε in the oligosaccharide moieties of the two forms. Treatment of the radiolabeled secreted enzyme with various endoglycosidases revealed the preεence of three N- linked oligoεaccharide chains, two high-mannoεe typeε (Endo H εensitive) and one complex type, the latter being Endo H and F resistant. Analyses of the Endo H- releaεed oligosaccharides revealed that one had two phosphate residues which specifically bound to immobilized mannose-6-phosphate receptors while the other was a hybrid structure containing sialic acid. These physical and kinetic properties and the presence of complex-type oligosaccharide chains on the recombinant secreted enzyme were similar to those of 0 the native enzyme purified from human plasma. The secreted form of α-Gal A was taken up by cultured Fabry fibroblaεts by a saturable process that waε blocked in the preεence of 2 mM mannoεe-6-phosphate indicating that binding and internalization were 5 mediated by the mannose-6-phosphate receptor. The binding profiles of the recombinant secreted enzyme and the α-Gal A secreted by NH 4 Cl-treated human fibroblastε to the immobilized receptor were identical. The production of large amounts of o soluble, active recombinant α-Gal A in accordance with the invention, which is similar in structure to the native enzyme isolated from plasma, will permit further comparison to the native enzyme formε and the clinical evaluation of enzyme replacement in Fabry 5 diεeaεe. 7 . 1. MATERIALS AND METHODS 7 . 1. 1. MATERIALS 0 Endo-7-N-acetylglucosaminidaεe H (Endo H) , endo- β-N-acetylglucoεaminidaεe D (Endo D) , endoglycoεidaεe F (Endo F) and peptide:N-glycoεidaεe F (PNGaεe F) were obtained from Boeringer Mannheim, Indianapolis, IN. [ 35 S]-methionine (>1,000 Ci/mmol) , D-[2,6- 3 H)-mannose 5 (60 Ci/mmol) , 32 P-Phosphoruε (10 mCi/ml) and Amplify were obtained from Amersham, Arlington Heights, IL. Pansorbin was obtained from Calbiochem, San Diego, CA. 4-MU glycosides were obtained from Genzyme, Cambridge, MA. Freund's adjuvants, sphingomyelin (from brain) and phenylmethylsulfonyl fluoride (PMSF) were obtained from Sigma, St. Louiε, MO. QAE Sephadex, Sephadex G- 25, octyl Sepharoεe and Superoεe 6 were obtained from Pharmacia-LKB, Piscataway, NJ. The TLC silica plates (cat. 5626) were purchased from EM Science, Gibbstown, NJ. The COS-l cell line was obtained from the ATCC. All tisεue culture reagentε were obtained from Gibco, Grand Island, NY. Sinti Verse I scintillation cocktail was obtained from Fisher, Pittsburgh, PA. The immobilized mannose-6-phoεphate receptor waε obtained from Dr. Stuart Kornfeld, Waεhington Univerεity, St. Louis, MO. The pyrene-dodecanoyl- sphingosyl-trihexoside (P-C, 2 STH) was obtained from Dr. Shimon Gatt, Hebrew University, Israel. Apolipoprotein E was obtained from BTG Inc. , Ness- Ziona, Israel. 7.1.2. CELL CULTURE Cells were maintained at 37"C in 5% C0 2 in Dulbecco's Modification of Eagle's Medium (DMEM) with 10% fetal calf serum (FCS) and antibiotics. The DG44 line was cultured in DMEM supplemented with HT (hypoxanthine, thymidine, Sigma) while the recombinant CHO line DG5.3 received DMEM supplemented with 10% dialyzed FCS. (Kaufman, et al. , 1988, J. Biol. Chem. 263: 6352). 7.1.3. PURIFICATION OF RECOMBINANT α-Gal A Recombinant CHO culture media was collected (20 L) and concentrated to 500 ml using a Pellicon cassette tangential-flow concentrator, with a molecular weight cutoff of 10,000 daltons (Millipore, MA). The pH of the concentrate was adjuεted to 4.7 to 5.0 with 10 N HCl and εubsequently clarified by centrifugation at 10,000 x g in an RC-5 refrigerated centrifuge for 10 min. All chromatographic stepε were automated on an FPLC apparatuε (Pharmacia) and were performed at room temperature. Approximately 100 ml of the media concentrate (-20 mg of α-Gal A enzyme protein) was applied to an α-Gal A affinity column (α-GalNH 2 - Sepharose; 2.5 x 8 cm) (Bishop & Desnick, 1981, J. Biol. Chem. 256: 1307) pre-equilibrated with buffer A (0.1 M citrate-phosphate, pH 4.7, 0.15 M NaCl). The column was washed with buffer A until the protein concentration in the eluate returned to the pre- application level (-200 ml) and was eluted with 150 ml of buffer B (0.1 M citrate-phosphate, pH 6.0, 0.15 M NaCl, 70.4 mM galactose). The eluate was collected, concentrated to about 20 ml using an ultrafiltration cell, molecular weight cutoff 30,000 daltons, under poεitive nitrogen pressure (Amicon) . The concentrate was mixed with an equal volume of buffer C (25 mM Bis- Triε, pH 6.0, 3 M (NH 4 ) 2 S0 4 ) , centrifuged at 10,000 x g and the pellet which contained up to 40% of the activity, was redissolved in buffer A and mixed with an equal volume of buffer C and centrifuged as above. The combined supernatants were applied to a column of Octyl-Sepharose (1.5 x 18 cm) pre-equilibrated with buffer D (25 mM Bis-Tris, pH 6.0, 1.5 M (NH 4 ) 2 S0 4 ) . The column was washed as above until the eluting protein concentration returned to pre-application levels (-100 ml) and the column was eluted with buffer E (5 mM sodium-phosphate, pH 6.0, 50% ethylene glycol). The product from three Octyl-Sepharose elutions, totalling approximately 75 ml, was concentrated as above to about 2 ml using an Amicon concentrator. The concentrate was finally applied to a column of Superose 6 (20-40 μm, Pharmacia, 1.6 x 100 cm) equilibrated in buffer F (25 mM sodium phosphate, pH 6.5, 0.1 M NaCl). The α-Gal A peak was collected, -20 ml, concentrated as above and stored in buffer F at 4°C. 7-1.4. ENZYME AND PROTEIN ASSAYS α-Gal A was asεayed uεing 4-methylumbelliferyl-α- D—galactopyranoεide (4-MU-α-Gal) as previously described (Bishop, et al., 1980, In Enzyme Therapy in Genetic Diseaseε: 2. Desnick, R.J. (Ed.). Alan R. Liεs, Inc. New York, p. 17). Briefly, a stock solution of 5 mM 4-MU was prepared in 0.1 M citrate- phosphate buffer, pH 4.6 solubilized in an ultrasonic bath. The reaction mixtures containing 10-50 μl of enzyme preparation or cell extracts and 150 μl substrate, were incubated at 37°C for 10-30 min. The reactions were terminated with the addition of 2.33 ml of 0.1 M ethylenediamine. One unit of activity is that amount of enzyme which hydrolyzes 1 nmol of εubεtrate/hour. Endo H, Endo D, Endo F and PNGaεe F digeεtions were performed as deεcribed (Tarentino, et al., 1989, Meth. Cell. Biol. 32: 111) . Sampleε were diluted to 0.2-0.5% SDS before digestion. All reaction volumes were 50 μl. A drop of toluene was added to each reaction tube to prevent bacterial growth. Briefly, Endo H digestionε (5 mU/reaction) were performed at 37°C overnight in 5 mM εodium citrate, pH 5.5 and 0.2 mM PMSF. Endo D digeεtionε (10 mU/reaction) were performed at 37°C overnight in 0.2 M citrate phosphate buffer, pH 6.0 and 0.2 mM PMSF. Endo F digestions (50 mU/reaction) were performed overnight at 30°C in 0.17 M sodium acetate, pH 6.0, 1.6% NP-40 and 0.2 mM PMSF. PNGase F digestions (100 mU/reaction) were carried out overnight at 30°C in 0.17 M potasεium phosphate, pH 8.6, 1.6% NP-40, 0.2 mM PMSF. Protein concentration was determined by the fluorescamine method (Bohlen, et al., 1973, Arch. Biochem. Biophys. 155: 213) as modified by Bishop et al. (Bishop et al., 1978, Biochim. Biophys. Acta 524: 109) . ' 7.1.5. IN VIVO NATURAL SUBSTRATE ASSAY For this asεay, 30 nmoleε of P-C 12 STH and 70 nmoles of sphingomyelin were mixed in a chloroform:methanol solution (1:1), evaporated under nitrogen and dried in a Speed-Vac (Savant) . The pellet was resuspended in 2 ml of saline, sonicated using a Heat Systems Ultrasonicε, Inc., Microson sonicator for 3-5 min at 40% output power and allowed to stand at room temperature for 1 hour. Apolipoprotein E (80 μg) was added and the mixture was incubated for an additional 15 min at room temperature. The liposomeε were added to the culture media of recombinant CHO cellε and incubated at 37"C in a C0 2 incubator for 1 to 4 hourε. Cells were removed from the culture disheε by trypεinization, washed once in DMEM supplemented with 10% fetal calf serum and twice with saline. The cell pellet was resuspended in chloroform-methanol and heated to 60"C for 10 min and centrifuged at 600 x g for 10 min. The supernatant was dried under nitrogen and the pellet resuspended in 100 μl of chloroform:methanol. Samples were spotted on a silica gel thin layer chromatography plate and chromatographed in chloroform:methanol:water (90:10:1) for 45 min followed by chromatography in chloroform:methanol:water (75:25:4) for 30 min. Products were visualized under UV light (330 nm) , exciεed from the plate by εcraping, resuspended in chloroform:methanol, and their fluorescence quantitated in a Farrand spectrofluoremeter (343 nm excitation, 378 nra e isεion) . 7.1.6. POLYCLONAL ANTIBODIES A New Zealand white rabbit (2 kg) waε injected with 150 μg of purified splenic α-Gal A in Freund's complete adjuvant prepared as follows: 150 μg of α- Gal A was added to 0.5 ml of PBS in a glasε syringe. Using a stainleεε steel 21 guage needle, the PBS/α-Gal A solution was mixed with 0.5 ml of Freund's complete adjuvant in a second glasε εyringe, until a homogenouε emulsion was obtained. The emulsion was injected into 8 different subcutaneous sites (back) and 1 intramuscular site (thigh) . Two months following the initial injection, the rabbit was booεted with 50 μg of α-Gal A in Freund'ε incomplete adjuvant aε above. Serum waε collected from an ear vein at dayε 8 and 12 following the booεt. The titer waε checked using a standard ELISA asεay (Johnεtone & Thorpe, 1982, Iummunochemistry in Practice. Balckwell Scientific Publications, Oxford) . Subsequent boostε were given approximately every two monthε followed by a bleeding 10 dayε later. A typical bleed yielded 30-40 ml of blood. 7.1.7. SDS-PAGE AND AUTORADIOGRAPHY Polyacrylamide gel electrophoreεis was carried out under reducing conditions (where appropriate) as described by Laemmli in a 1.5 mm thick slab containing 10% acrylamide (Laemmli, U.K., 1970, Nature 227: 680). The gel was fixed in 10% acetic acid and 20% methanol for 30 min and then soaked in Amplify for 30 min with agitation. Gels were vaccum dried for 90 min (Hoffer) and exposed to Kodak X Omat AR for 4 to 72 hours. 7.1.8. ISOELECTRIC POINT AND H OPTIMUM DETERMINATION The isoelectric point was determined using QAE sephadex essentially as described by Yang and Langer (Yang & Langer, 1987, Biotechniques 5: 1138). The pH optimum was determined in 25 mM sodium phosphate 0 buffer at 37°C. 7.1.9. MANN0SE-6-PH0SPHATE RECEPTOR AFFINITY CHROMATOGRAPHY AND QAE SEPHADEX CHROMATOGRAPHY 5 The 215 kDa mannose-6-phosphate receptor (M-6-P receptor) coupled to Affigel-10 was at a concentration of 0.4 mg/ml of packed gel. Samples, in binding buffer (50 mM imidazole, pH 7.0, 150 mM NaCl, 0.05% Triton X-100, 5 mM sodium-3-glycerolphosphate, 0.02% o sodium azide), were applied to a 1.5 x 0.8 cm column at a flow rate of 0.3 ml/minute. Following sample application (5 ml) , the column was washed with 5 ml of binding buffer and eluted with a nonlinear gradient of mannose-6-phosphate in binding buffer (0-5 mM) . Thiε 5 exponential gradient (Dong, et al., 1990, J. Biol. Chem. 265: 4210) was formed by an apparatus consisting of two chambers of 2.5 cm diameter and 1 cm diameter. Fractions were collected (0.5 ml) and 10 μl aliquots assayed for α-Gal A activity using 4-MU-α-Gal, and for 0 radioactivity using 10 ml of Sinti Verse I scintillation coctail. QAE Sephadex chromatography in a 3 x 0.8 cm column was performed as described (Varki & Kornfeld, 1983, J. Biol. Chem. 258: 2808; Varki & Kornfeld, 5 1980, J. Biol. Chem. 255: 10847) . Briefly, following digestion with Endo H, the released oligosaccharides (labeled with [ 3 H]-mannose) were isolated and desalted on an 18 x 0.8 cm column of Sephadex G-25. Samples were applied to the column of QAE Sephadex and eluted with εuccessive 5 ml aliquots of 2 mM Triε, pH 8.0 containing 0, 20, 40, 80, 100, 120, 140, 160, 200, 400 and 1,000 mM NaCl. Oligosaccharideε eluted according to the number of their negative chargeε; 0 charge at 0 mM NaCl, 1 at 20 mM NaCl, 2 at 70 mM NaCl, 3 at 100 mM NaCl and 4 at 140 mM NaCl. 7.1.10. LABELING OF CELLS WITH [ 35 S]-METHIONINE, T 3 H1-MANNOSE AND f 32 Pl-PHOSPHOROUS Confluent cultureε in 100 mm diεhes were washed once with 5 ml of methionine-free DMEM. A fresh aliquot of this medium (5 ml) was placed in each dish and cultureε were incubated in a 37"C incubator for 30 min. The media was removed from the disheε and a freεh aliquot of methionine-free DMEM (1 ml) , εupplemented with 10% dialyzed FCS and 50-100 μCi of [ 3S S]-methionine was added. Cellε were incubated at 37°C for 3 to 5 min, the radioactive media was removed and cells washed twice with DMEM pluε FCS. Cellε were chaεed for the indicated timeε in 5 ml of DMEM plus FCS containing 2 mM methionine. For overnight labeling, cultures received 5 ml of methionine-free DMEM supplemented with dialyzed FCS, glutamine, antibiotics, 10 mM NH 4 C1 and 200 μCi [ 35 S]-methionine. For [ 3 HJ-mannoεe labeling, cultureε were grown as above in supplemented DMEM. Cells were waεhed with 5 ml of low-glucoεe DMEM and a freεh aliquot of media waε added. [ 3 H]-mannose (250 μCi; dried under nitrogen and resuspended in DMEM) , was added and cells were incubated in a 37°C incubator for 24 hourε. For 3 P labeling, cultureε were εwitched to phoεphate-free DMEM εupplemented with 10% dialyzed FCS . Following addition of [ 32 P ] -orthophosphate ( 1 mCi ) cultures were incubated in a 37 °C , C0 2 incubator for 24 hours . 7.1.11. CELL LYSIS AND IMMUNOPRECIPITATION Cells grown in 100 mm culture diεheε were waεhed twice with 5 ml of phoεphate buffered εaline (PBS) and scraped into 12 ml conical tubes using a rubber policeman and 10 ml of PBS. Following centrifugation at 2,500 rpm for 10 min cells were resuspended in 1 ml of 25 mM NaP0 4 , pH 6.0 and received three 15-second bursts in a Branson cup sonicator. Cell debris was removed by centrifugation (10,000 x g for 15 min at 4°C) . Alternatively, cells were washed as above and 1 ml of lysis buffer (50 mM sodium phosphate, pH 6.5, 150 mM NaCl, ImM EDTA, 1% NP-40, 0.2 mM PMSF) was added to the dish. The culture dish was incubated at 4°C for 30 min and cells were transferred to a 1.5 ml microcentrifuge tube. Cell debris was removed as above. Immunoprecipitation was carried out as described (Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory Press pp. 18.42-18.46) . Briefly, 0.5 ml of cell lysate or culture media was placed in a 1.5 ml microcentrifuge tube and 50 μl of preim une rabbit εerum waε added. The mixture waε incubated at 4°C for 1 hour with gentle agitation. Fifty μl of Pansorbin was added and incubation was continued for 30 min. The mixture waε clarified by centrifigation at 10,000 x g for 5 min, 100 μl of anti-α-Gal A polyclonal antibody was added and incubation was continued for 1 hour at 4°C with gentle rocking. Pansorbin (100 μl) was added and incubation continued for 30 min as above. The tertiary S ^ _ aureus cells-antibody-antigen complex was collected by centrifugation as above. The supernatant was discarded and the pellet washed successively in NET buffer (50 mM sodium phosphate, pH 6.5, 150 mM NaCl, 1 M EDTA, 1% NP-40, 0.25% gelatin) supplemented with 0.5 M NaCl, in NET buffer with 0.1% SDS and in TN buffer (10 mM Tris, pH 7.5, 0.1% NP-40) . The immunoprecipitated protein was denatured by heating at 100°C for 5 min in the presence of 2% SDS, 100 mM DTT 0 (DTT was not used for experiments involving secondary structure conformationε) . S_;_ aureus cellε were removed by centrifugation at 10,000 x g for 5 min at room temperature. 5 7.2. RESULTS 7.2.1. PURIFICATION Recombinant α-Gal A produced in the cell bioreactor waε purified from the crude media by o affinty chromatography on α-GalNH 2 -C 12 -Sepharoεe (Bishop & Desnick, 1981, J. Biol. Chem. 256: 1307) followed by hydrophobic chromatography on Octyl Sepharose and gel filtration on a 100 cm Superose 6 column as deεcribed above. Table VI εhows a typical purification of a 20 5 mg lot of recombinant α-Gal A and the εpecific activitieε of the enzyme at each εtage of purification. The recombinant enzyme waε eεsentially homogeneous, following the gel filtration step, and was >98% pure as judged by SDS-PAGE (FIG. 5) . A minor 0 contaminant of bovine serum albumin was removed by an additional gel filtration εtep on a column of Blue- Sepharoεe (Traviε, et al., 1976, Biochem. J. 157: 301) reεulting in an enzyme preparation which was greater than 99% pure as judged by loading 20 μg of α-Gal A on 5 SDS-PAGE. TABLE VI FPLC Purification Of Recombinant α-GAL A A Typical Purification Run Starting With 20 MG Of α-GAL A Step ϋ x l0 ] U x 10 3 Fold Yield Purification % That the purification of recombinant α-Gal A would be facilitated by growth of the CHO cellε in serum-free media was demonstrated by metabolic labelling of total cellular and secreted protein. In contrast to the result seen in FIG. 5, radiolabeled α- Gal A was eεεentially the only protein seen in the media of the high-expresεor line, DG5.3 (FIG. 6). 7.2.2. PHYSICOKINETIC PROPERTIES Recombinant α-Gal A was found to have a subunit molecular weight of -57 Kd based on SDS-PAGE (FIG. 5) . The Km towards the artificial substrate 4-MU-α-D— galactopyranoside was 1.9 mM (FIG. 7A) and the pH optimum and isoelectric point were 4.6 and 3.7 reεpectively (FIG. 7B and 7C) . In order to determine whether the recombinant enzyme recognized and hydrolyzed itε natural εubεtrate, liposomes containing the fluorescently- labeled α-Gal A substrate P-C I2 STH and apolipoprotein E (for lysosomal targeting) were incubated with CHO cells over-expressing α-Gal A (clone DG5.3) . As εhown in FIG. 8A-8B, recombinant lyεosomal α-Gal A rapidly hydrolyzed the substrate to P-C )2 SDH (the dihexoside) . The rapid hydrolysis of P-C 12 STH indicates that recombinant α-Gal A can recognize this natural substrate analog and very efficiently hydrolyze it. Also, since this subεtrate iε targeted to the lysoεome, cell associated recombinant α-Gal A must be correctly targeted to thiε location. Theεe reεultε indicate that recombinant α-Gal A produced and εecreted by CHO cellε is essentially identical to the enzyme purified from human plasma (Table VII) . TABLE VII Property Comparison Of Recombinant α-GALACTOSIDASE A Enzyme Purified From Human Tissue α-Gal A 7.2.3. PROCESSING AND RATE OF SECRETION OF RECOMBINANT α-Gal A Nascent polypeptides, transversing the endoplasmic reticulum assume secondary structure conformations cotranslationally or soon after their synthesis is completed (Gething, et al., 1989, Meth. Cell. Biol. 32: 185). α-Gal A was labeled with [ 35 S]- methionine for three min and then chased with cold methionine for the indicated times. Immunoprecipitated α-Gal A was visualized on SDS-PAGE. The sampleε were prepared without DTT in order to maintain disulfide bridges that might have formed during the chase, indicative of a secondary structure conformation. A control (+DTT) was prepared by boiling an aliquot of the 60 minute sample in the presence of DTT to destroy disulfide bonds and the secondary structure. At 0 rain of chase (after 3 min of labeling) there was already a change in the mobility of this enzyme indicating that conformational changes occur cotranslationally or soon after completion of synthesis (FIG. 9) . Arrival of the new polypeptide to the Golgi network was detected by the acquisition of Endo H resiεtant oligoεaccharideε (Gething, et al., 1989, Meth. Cell. Biol. 32: 185). Radiolabeled α-Gal A (3 minute pulse) was chased with nonradioactive methionine and immunoprecipitated as above. The immunoprecipitates were then treated with Endo H and visualized on SDS-PAGE. Between 2 and 7 min of chase, the first Endo H-resistant form of α-Gal A could be detected, indicative of arrival of the recombinant enzyme at the Golgi, about 5 to 10 min following itε εyntheεiε (FIG. 10) . The majority of the Endo H εensitive form was rendered resistant by 60 min of chase. This enzyme transverses the Golgi network and is εecreted at 45 to 60 min of chase (FIG. 11) . Analysis of total media, from [ 35 S]-methionine labeled cells, revealed that >95% of the secreted protein by the recombinant CHO cells was α-Gal A (FIG. 12) . 7.2.4. ANALYSIS OF CARBOHYDRATE MOIETIES ON RECOMBINANT α-Gal A There are four N-glycosylation consensus sequences (Asn-X-Ser/Thr) in the α-Gal subunit predicted by the cDNA sequence. The fourth site iε probably not utilized εince it contains a proline residue in the X position. Recombinant α-Gal A was digested with Endo H, Endo F, Endo D and PNGase F. Digestion with PNGase F cauεed an ™7 kDa εhift in mobility on SDS-PAGE of half of the α-Gal A (FIG. 13). This change in molecular weight can be attributed to the removal of 3 N-linked carbohydrate moieties. Digestion of the recombinant enzyme with a cocktail of Endo H, Endo F and PNGase F did not result in any further decrease in molecular weight, indicating that all of the enzyme contains three N-linked carbohydrate moieties. Endo D, a glycosidaεe with a εtrict εpecificity for the lower Manαl-3 branch of the high-mannoεe core pentasaccharide (Tarentino, et al., 1989, Meth. Cell. Biol. 32: 111), did not have an effect on the mobility of α-Gal A, indicating that the recombinant enzyme does not contain this type of oligosaccharide (FIG. 13) . Endo H and Endo F together resulted in a 4 kDa shift indicating that two out of the three oligosaccharides on this enzyme are of the high- mannose type (Varki & Kornfeld, 1980, J. Biol. Chem. 225: 10847). Interestingly, intracellular α-Gal A was completely sensitive to PNGase F while half of the secreted enzyme was partially resistant to PNGase F (FIG. 14) . Since this resistance was eliminated by co-treatment with Endo H and Endo F (FIG. 13) , further 5 studies are necessary with Endo H and Endo F separately to determine the molecular nature of either the selective inhibition of PNGase F or the resistance of a proportion of the recombinant secreted enzyme to PNGase F digestion. 0 Having determined that the recombinant enzyme contains three oligosaccharideε, two of which are of the high-mannose type, the effect of inhibition of glycosylation was investigated (Furhmann, et al., 1985, Biochim. Biophys. Acta 825: 95) . Processing and 5 secretion of recombinant α-Gal A is not affected by selective inhibition of oligosaccharide processing. In the presence of deoxynojirimycin (an inhibitor of glucosidase I and II) , deoxymannojirimycin (an inhibitor of mannoεidaεe I) , and swainsonine (an o inhibitor of mannoεidaεe II) α-Gal A εecretion rate remainε the same aε the controls (FIG. 15) . However, tunicamycin (an inhibitor of oligosaccharide addition) inhibits εecretion of α-Gal A by aε much aε 80% (FIG. 15) . The εecreted enzyme from tunicamycin-treated 5 cultures could bind to a Con A Sepharose column indicating that thiε enzyme is partially glycosylated, probably due to incomplete inhibition of glycosylation by tunicamycin. These resultε indicate that oligosaccharide addition but not the processing events 0 tested is necessary for maturation and secretion of the recombinant enzyme. 7.2.5. PHOSPORYLATION Since the recombinant α-Gal A contained high- 5 mannose moieties, the recombinant enzyme could contain M-6-P and be competent for receptor mediated uptake. Cells from clone DG5.3 were metabolically labeled with [ 3 P]-orthophosphate for 12 hours and then the cell extracts and media immunoprecipitated and visualized on SDS-PAGE. As shown in FIG. 16, both cell- associated and secreted α-Gal A were phosphorylated, preεumably at their carbohydrate moietieε aε εuggeεted by the in vitro experimentε described above. 7.2.6. ANALYSIS OF ENDO H SENSITIVE OLIGOSACCHARIDES The high-mannose oligosaccharideε were removed by treating immunoprecipitated [ 3 H]-mannoεe labeled α-Gal A with Endo H. Theεe oligoεaccharideε were analyεed by chromatography on QAE Sephadex (Varki & Kornfeld, 1983, J. Biol. Chem. 258: 2808; and, Varki & Kornfeld, 1980, J. Biol. Chem. 255: 10847). Two major formε of theεe oligosaccharides were detected, a form with 2 negative charges and one with 4 negative charges (FIG. 17A) . The negative charge can be contributed by a phosphodiester moiety (-1) , a phosphomonoeεter moiety (-2) or εialic acid (-1) . Treatment of theεe sugars with dilute HCl did not shift the profile of any of the peaks indicating that there are no phosphodiester groups on these sugars (FIG. 17B) (Varki & Kornfeld, 1983, J. Biol. Chem. 258: 2808). Treatment with neuraminidase causeε a shift of the -2 peak resulting in two new peaks at 0 and -1 negative charges (FIG. 17C) . Therefore, the charge of the -2 peak iε contributed by sialic acid, most likely two moietieε. The resulting -1 peak following neuraminidaεe treatment iε probably a partial digeεtion of the -2 peak by the enzyme. Treatment of theεe oligoεaccharides with alkaline phosphatase cauεed a εhift of the -4 peak to 0 negative charge (FIG. 17D) . There was no effect on the -2 peak, indicating that the charge of the -4 peak iε contributed by two phosphomonoeεter bonds while the -2 peak does not contain any such bonds. Thus, it is evident from these reεultε that Endo H releases two types of high- mannose oligosaccharideε from recombinant α-Gal A, one containing εialic acid (poεsibly a hybrid oligosaccharide) and the other containing 2 phosphomonoester bonds (presumably as mannose-6- phoεphate) . To further confirm theεe findingε peakε -2 and -4 were chromatographed on an immobilized mannoεe-6- phoεphate receptor column (FIG. 18) . Although peak -4 interacted weakly with the receptor, it could be bound to the column and required the addition of mannose-6- phosphate for elution. A very weak interaction was observed between the receptor column and the -2 peak, suggesting that a portion of these hybrid oligosaccharides may contain M-6-P. The weak interaction of the high-mannose oligosaccharideε with the M-6-P receptor could be explained by the abεence of the protein core (Varki & Kornfeld, 1983, J. Biol. Chem. 258: 2808) . DG5.3 cells were labeled with [ 35 S]-methionine and the secretionε chromatographed on a column of immobilized mannose-6-phosphate receptor. Notably, the recombinant enzyme bound ε rongly to the column waε eluted specifically by the addition of 5 mM M-6-P (FIG. 19) . 7.2.7. INTERACTION OF α-Gal A WITH THE MANNOSE-6-PHOSPHATE RECEPTOR Since recombinant α-Gal A has been εhown to contain mannose-6-phosphate moietieε, it waε important to eεtabliεh whether this was also true for normal human α-Gal A. CHO proteins were labeled with r 3S Sl- L J methionine in the presence of NH 4 C1, to cause quantitative secretion of newly synthesized lysosomal enzymes (Dean, et al., 1984, Biochem. J. 217: 27) . The media was collected and chromatographed on a column of immobilized mannose-6-phosphate receptor. The column was eluted with a gradient of mannose-6- phoεphate aε described above. This elution protocol can separate lysosomal enzymes into low and high affinity receptor-binding ligands (Dong & Sahagian, 1990, J. Biol. Chem. 265: 4210). The recombinant enzyme co-eluted with the bulk of the lysosomal enzymes at an M-6-P concentration indicative of high affinity forms (FIG. 20A) . The same experiment was performed with εecretionε of MS914 (normal diploid human fibroblaεtε) cellε (FIG. 20B) and 293 cellε (human adenovirus transformed embryonic kidney cellε) (FIG. 20C) . When the εame M-6-P gradient waε applied, human α-Gal A alεo co-eluted with the bulk of the lyεoεomal enzymeε, demonεtrating that the recombinant enzyme exhibitε affinity to the M-6-P receptor εimilar to that of the normal human enzyme. 7.2.8. RECEPTOR MEDIATED UPTAKE OF RECOMBINANT α-Gal A IN FABRY FIBROBLASTS Fabry fibroblasts were incubated with varying amounts of the recombinant enzyme for 6 hours (FIG. 21) . The enzyme uptake was εaturatable and was specifically inhibited by the addition of 2 mM M-6-P in the uptake media, indicating that the uptake was via the cell surface M-6-P receptor. 8. EXAMPLE: α-Gal A-PROTEIN A FUSION EXPRESSED IN MAMMALIAN CELLS The subεections below describe a fusion construct of the human α-Galactosidase A cDNA and the εtaphylococcal protein A IgG binding domain E expreεεed in COS-l cells and purified to apparent homogeneity by IgG affinity chromatography. The fuεion construct was engineered using PCR techniques to insert the 16 nucleotide collagenase cleavage recognition sequence between the α-Gal A and the protein A domain E sequence. In addition, the termination codon was deleted from the α-Gal cDNA and inεerted at the terminuε of the domain E εequence. Tranεient expreεεion of the fusion construct in COS-l cells resulted in a 6 to 7-fold increase over endogenous levels of α-Gal A activity and εignificant εecretion into the media (4,000 unitε; nmoleε/hour) . The fuεion protein from the culture media was purified to homogeneity on IgG sepharoεe chromatography. After collagenaεe treatment, the liberated α-Gal A was separated from the protein A peptide by IgG chromatography. By this method over 85% of secreted α- Gal A fusion protein waε purified aε the active, glycoεylated homodimeric protein. Thiε method should be useful for the expresεion and rapid purification of normal and mutant proteinε. In addition, thiε conεtruct haε been inεerted into the CHO DG44 cellε εo that large amountε of the secreted recombinant enzyme can be produced and rapidly and efficiently purified. 8.1. MATERIALS AND METHODS 8.1.1. MATERIALS Restriction endonucleases, Taq polymeraεe, T4 ligaεe and pGem plaεmidε were obtained from Promega (Madiεon, WI) . Vector pRIT5 and IgG-Sepharoεe were purchaεed from Pharmacia (Piscataway, NJ) . Sequenase εequencing kitε were from United States Biochemical Corp. (Cleveland, OH) . Collagenase was obtained from Sigma (St. Louis, MO). Oligonucleotides were synthesized using an Applied Biosystems DNA synthesizer model 380B. 8.1.2. CELL CULTURE AND TRANSFECTIONS COS-l cells were obtained from the ATCC (Rockville, MD) . The cells were cultured by standard techniques in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal calf serum and antibiotics. Exponentially growing COS-l cells (5 x 10 6 cellε /T75 flaεk) were detached from the plastic by trypsinization, collected by centrifigation at 3,000 x g, and then washed once in ice-cold electroporation buffer (phosphate buffered sucrose: 272 mM sucrose, 7 mM sodium phosphate, pH 7.4, containing 1 mM MgCl 2 ) . Following centrifugation at 3,000 x g, the cells were resuεpended in 0.8 ml of electroporation buffer and placed in an electroporation cuvette with a 0.4 cm gap. Ten to fifteen μg of plaεmid DNA was added and cells were kept on ice for 5 min. The cell-containing cuvette waε placed in a Gene Pulεer electroporation apparatuε (Bio-Rad) and the cellε were pulsed at 350 V, 25 μF. The cells were maintained on ice for an additional 10 min and then placed into a 100 mm culture dish containing 10 ml of growth medium. 8.1.3. PCR, DNA SEQUENCING AND VECTOR CONSTRUCTIONS The fusion construct was synthesized using a recently described PCR technique (Ho, et al., 1989, Gene 77: 51; Kadowaki, et al. , 1989, Gene- 76: 161). Briefly, the full-length α-Gal A cDNA waε εubcloned into the pGEM plaεmid and the reεulting pG6-AGA plaεmid waε uεed for PCR amplification of the α-Gal A sequence with primers designed to delete the termination codon, to add a collagenase cleavage conεenεus sequence at the 3 ' end and to include an Eco RI recognition εequence at the 5 ' end of the cDNA (FIG. 22) . The sense primer was 5'- CCGAATTCATGCTGTCCGGTCACCGTG-3 ' and the antisense primer was 5 '-CGCCGGACCAGCCGGAAGTAAGTCTTTTAATG-3 ' . The protein A domain E (Nilsεon, et al., 1985, EMBO J. 4: 1075) waε εimilarly amplified with the collagenaεe consensus sequence in the 5' oligonucleotide; the senεe and antiεenεe oligonucleotideε were 5'- CCGGCTGGTCCGGCGCAACACGATGAAGCT-3 ' and 5'- GGCCGAATTCCGGGATCCTTATTTTGGAGCTTGAGA-3 ' , reεpectively. The 1323 nt and 201 nt products of the α-Gal A and protein A PCR reactions were gel-purified on an 0.8% agarose gel and mixed together for the fuεion PCR reaction. The sense primer from the α-Gal A reaction and the antisenεe primer from the protein A reaction were uεed for the final fuεion reaction. The product of thiε reaction waε digeεted with Eco RI and ligated into the Eco RI digested plasmid pGEM4Z. The protein A domain E and junctions between the linker and α-Gal A and protein A were confirmed by the dideoxynucleotide chain termination sequencing method of Sanger (Hanahan & Meselεon, 1985, Methods Enzymol. 100: 333) . The confirmed fuεion εequence then was digeεted with Eco RI and subcloned into the eukaryotic expreεsion vector p91023(B). 8.2. RESULTS 8.2.1. CONSTRUCTION OF α-Gal A-PROTEIN A (AGA-PA) FUSION FIGURE 22 showε the εtrategy uεed for the conεtruction of the α-Gal A-Protein A domain E fuεion sequence. The full-length α-Gal A cDNA (1323 nt) and protein A domain E sequence (201 nt) were amplified separately and then fused by a second PCR amplification (FIG. 22) using the 5' α-Gal A cDNA εense primer (PI) and the 3' Protein A antisense primer (P4). The primers were designed to (a) eliminate the α-Gal A TAA stop codon; (b) insert the 16 nt collagenase cleavage consensus recognition sequence encoding Pro Ala Gly Pro between the α-Gal A and protein A cDNA sequence; and (c) introduce a TAA stop codon at the 3' end of protein A domain E. The integrity of thiε construct was confirmed by sequencing the protein A domain, linker and 3' of the α-Gal A cDNA (FIG. 23). 8.2.2. EXPRESSION OF pAGA-PA IN COS-l CELLS Seventy-two hours after transfection with the pAGA-PA construct, maximal levels of 4MU α-Gal activity were detected in cell extracts and in the spent culture media (Table VIII) . TABLE VIII Transient Expresεion Of AGA-PA Conεtruct In COS-l Cellε. Following Tranεfection A 7-Fold Increase In Endogenous α-Gal A Activity Was Observed. Also, An Increaεe Of α-Gal A In The Culture Media Waε Obεerved α-Gal A Activity* COS-l CELLS Cellε Media Control Tranεfected *Aεεayed uεing 4MU-α-Gal aε εubεtrate. Compared to the endogenous α-Gal A activity in COS-l cells of 210 U/mg, the transfected cells expresεed 1300 U/mg. No α-Gal A activity was detected in the spent culture medium of uninfected COS-l cells whereas 72 hours after transfection, 400 units of activity were secreted into the media. 8.2.3. AFFINITY PURIFICATION OF α-Gal A The spent media from a single 100 mm dish of COS- 1 cells was collected 72 hourε after tranεection and applied to a column of IgG-Sepharoεe. Minimal activty of α-Gal A paεεed through the column during sample application (flow-through) , or during the buffer wash (Table IX) . However, more than 95% of the bound α-Gal A fuεion protein waε eluted by the addition of 0.5 M acetic acid (elution buffer) . TABLE IX IgG Sepharose Chromatography Of The α-Gal A Protein A Fusion Product From The Culture Media Of Tranεfected COS-l Cellε Purification α-Gal A Activity* Step fU/ml) Medium 4,400 Flow-Through 10 Buffer Waεh 0 Elution** 4,200 * Ten ml of culture media were applied to the column, waεhed and eluted aε described in "Methods". α-Gal A activity was aεεayed uεing 4MU-α-Gal as εubstrate. ** 0.5 M HAc, pH 3.4 8.2.4. RELEASE OF THE PROTEIN A DOMAIN FROM THE AGA-PA FUSION PROTEIN The affinity purified fusion protein was treated with collagenase for 1 hour and the reaction products were rechromatographed on the IgG affinity column (Table X) . The Protein A domain E was readily bound to the IgG column, whereas the human α-Gal A waε eluted in the flow-through. Almost 90% of the applied activity was eluted. Based on the specific activity of the purified enzyme, it waε eεtimated that thiε procedure resulted in 90% pure enzyme. TABLE X Treatment Of α-Gal A-Protein Fusion With Collagenase. Upon Treatment The Bindng Of The IgG Column Decreased From 69% To 11%. % Of Recovered α-Gal A Activity* STEP COLLAGENASE** + Flow-Through 31 89 Elution 69 11 * Assayed using 4MU-α-Gal as substrate; a total of 4,200 units of α-Gal A activity was applied. ** Collagenase treatment for 1 hour at 25 "C . 9. EXAMPLE: IN VIVO MODIFICATION OF RECOMBINANT HUMAN α-Gal A GLYCOSYLATION BY α 2 . 6-SIALYLTRANSFERASE The example presented here describes a method whereby recombinant human α-Gal A enzyme is produced in a form that more closely resembles the native plaεma glycoform of the enzyme. Specifically, human α-Gal A is produced in CHO cell lines that have been engineered to contain an α-2,6-sialyltranεferase gene such that the α-Gal A protein made in these cell lines is sialylated. Results presented below demonstrate that the biologically active α-Gal A enzyme produced using such cell lines exhibit a broader tisεue distribution, which can enhance this recombinant α-Gal A'ε therapeutic value. 9.1. MATERIALS AND METHODS 9.1.1. CONSTRUCTION OF THE α2,6- SIALYLTRANSFERASE EXPRESSION VECTOR, PST26 The 1.6 kb rat CDNA (ST3) encoding the complete amino acid εequence of the 3-galactoεide α2,6-sialyl- tranεferaεe (Gal α2,6ST; Weinεtein et al.. 1987, J. Biol. Chem. 262: 17735) was εubcloned into the Bam HI εite of the mammalian expression vector pRLDN, a gift from Smith, Kline and French Laboratories, resulting in the vector designated pST26. This construct was introduced by electroporation into the CHO cell line DG5.3-1000MX, a high overexpreεεor of human α- galactoεidaεe A. Clones were εelected by growth in media containing 500 μg/ml of G418 to select for expression of the pST26 neo gene. Positive cloneε were individually isolated using cloning rings and then each was analyzed for expresεion of total εecreted recombinant human α-galactosidase A. In addition, the percent of the secreted recombinant α- galactosidase A that contained α2,6-sialic acid residues was determined by Sa bucus nigra agglutinin (SNA) chromatography on a column of SNA-Sepharose as described by Lee et al. (1989, J. Biol. Chem. 264:13848-13855) . 9.1.2. SNA-LECTIN FLUORESCENCE MICROSCOPY For fluorescence microscopy, positive DG5.3- 1000MX-ST26 clones were grown on 12-ram glass coverslipε and then εtained with fluoreεcein iεothiocyanate (FITC)-tagged SNA aε previouεly deεcribed (Lee et al. , 1989, J. Biol. Chem. 264:13848- 13855) . Briefly, the cellε were fixed with a fresh solution of 2% para-formaldehyde, 0.1% glutaraldehyde in phosphate buffered εaline (PBS), pH 7.0, for 1 hr at 23°C, and subsequently blocked with 50 mM NH 4 C1 in PBS for 30 min at 23 C C. Cells were incubated with FITC-SNA at a concentration of 25 mg/ml in PBS for 30 m i n at 23°C. Following washing in PBS, the coverslips were mounted in 15% vinol 205 polyvinol alcohol, 33% glycerol, 0.1% εodiura azide in 100 mM Triε, pH 8.5. The cellε were viewed with a Zeiεε Photomat III fluoreεcence microεcope and were photographed uεing Kodak Gold color film, ASA 25. 9.1.3. PURIFICATION OF RADIOLABELLED HUMAN α-GAL A WITH AND WITHOUT α2,6-SIALIC ACID RESIDUES Parental DG5.3-1000MX cells and modified DG5.3- ST26.1 cells were grown in 1 liter roller bottles ( _ 5 x 10 8 cells) with 125 ml of DMEM, containing 10% dialyzed fetal calf εerum (GIBCO, Grand Iεland, NY) and 500 μCi[ 35 S]-methionine. The radioactive medium waε replaced daily for three dayε, and the εpent medium from each line was collected, pooled and concentrated using a stirred cell concentrator (Amicon, Beverly, MA) . Approximately 2 mg (4 x 10 6 U) of recombinant enzyme secreted from the DG5.3-l000Mx and DG5.31000MX-ST26.1 cell lines, respectively, was individually purified to a specific activity of 100 cpm/U of enzyme. The purified, radiolabeled, secreted recombinant α-galactosidase A preparations (0.5 mg each) were treated individually with 5 units of acid phosphataεe (Sigma) and the dephoεphorylated forms also were used for intravenous injections. 9.1.4. IN VIVO HALF-LIFE AND TISSUE DISTRIBUTION OF RECOMBINANT α-GAL A For the plasma half-life determinations, each radiolabelled α-Gal A form was injected into the tail vein of two CDI female mice. Each animal was bled through the sinus plexus at timed intervals over a 10- 20 min period. Blood samples were centrifuged in an IEC Microhematocrit centrifuge and 50 μl of plasma was added to an aqueous scintillation fluid (AquaSol, NEN, Boεton, MA) for counting. To assesε the tiεεue diεtribution of the different enzyme forms, two mice for each enzyme were sacrificed by decapitation 4 hr after enzyme injection, selected tissues were removed, and the radioactivity in various tissues was deter¬ mined and expresεed per gram of tiεεue wet weight. Each value iε the average of two independent experimentε. 9.2. RESULTS 9.2.1. INTRODUCTION OF PST26 INTO DG5.3-1000MX CHO CELLS AND DEMONSTRATION OF α2,6-SIALYLTRANSFERASE ACTIVITY IN G418-SELECTED CLONES Following electroporation of the pST26 plaεmid into CHO DG5.3-1000MX cells, transformed cells were selected which were resistant to the antibiotic G418. From the pool of resistant cells, 10 individual clones were isolated and expanded for further studieε. These clones were designated DG5.3-1000MX-ST26.1 to ST26.10. As evidence for the presence of α2, 6-sialyltransferase activity in these cellε, each cell line was stained with FITC-SNA and then examined by phase contraεt and fluoreεcence microεcopy. All ten of the DG5.3-lOOOMx- ST26 cell lines were positively stained by the fluorescent lectin, indicating that various cell membrane glycoproteins εerved aε acceptorε for the expreεεed α2,6-sialyltransferase. In contrast, the FITC-lectin did not stain the parental DG5.3-1000MX cells. 9.2.2. CHARACTERIZATION OF RECOMBINANT HUMAN SECRETED α2.6-SIALYLATED α-GAL A Each of the ten DG5.3-1000MX-ST26 cell lines and the parental DG5.3-1000MX cellε were grown in 100 mm diεhes at the same cell denεity. The amount of εecreted human α-Gal A activity per mg protein of total cultured cellε was determined for each cell line. The DG5.3-1000MX-ST26 lines secreted from 28 to 100% of the α-Gal A secreted by the parental cell line (16,000 U/mg protein, Table XI). To determine the percent of the recombinant human secreted α-Gal A that contained α2,6-sialic acid reεidueε, the secreted enzyme was chromatographed on the immobilized SNA column, and the bound enzyme was eluted with 0.4 M lactose. As shown in Table XI, the individual clones had 55 to 100% of the applied enzymatic activity that was specifically bound and eluted from the lectin column. Notably, clone DG5.3- lOOOMx-ST.l produced the higheεt amount of recombinant α-Gal A, eεsentially all the enzyme form was sialylated and bound the immobilized SNA column. In contrast, recombinant α-Gal A secreted by the parental DG5.3-1000MX cellε had little, if any binding to α2,6- εialic acid-εpecific lectin (Table XI) . TABLE XI DG5.3-1000MX-ST26 CHO Cell Lineε Secreting Human α2,6-Sialylated α-Galactosidase A Clone/ Percent of α-Gal A Subclone Secreted α-Gal A Secretion Bound to SNA-Lectin (%DG5.3-1000Mx * ) DG5.3-1000MX 5 100 DG5.3-1000MX-ST26 Subclones: 1 100 100 2 99 76 3 93 72 4 79 70 5 74 64 6 73 85 7 72 85 8 55 84 9 51 81 10 28 79 Activity iε expreεεed aε units per mg of cellular protein; The DG5.3-1000MX line secreted " 16,000 U/mg. 9.2.3. PLASMA HALF-LIFE AND TISSUE DISTRIBUTION OF α2 ,6-SIALYLATED HUMAN α-GAL A IN MICE To determine if the jLn vivo clearance of recombinant human α2,6-sialylated α-Gal A was different than that of the non-α2,6-sialylated enzyme, mice were injected with each form and their circulating half-liveε were determined. Aε εhown in Figure 24A, the presence of α2,6-sialic acid moieties increased the half-life of the enzyme in the circulation from 14 to almost 30 min. Further, treatment of the α2,6-sialylated and non-α2,6- sialylated recombinant enzymes with acid phosphatase increased the in vivo plasma half-life of the non- α2,6-εialylated enzyme (T 1/2 from 14 to 24 min), whereaε that of the phosphatase-treated α2,6-εialylated enzyme remained the same at about 28 min (Fig. 24B) . As depicted in Figure 25, the presence or absence of α2,6-εialic acid moietieε and/or phoεphate reεidueε, on the εecreted recombinant human α-Gal A forms had a marked effect on their reεpective tissue distributions following intravenous administration to mice. A greater percentage of the total α2,6- εialylated α-Gal A injected waε recovered in the lungs, kidney and heart, as compared to the non-α2,6- εialylated enzyme; in contraεt, leεε of the α2,6- εialylated enzyme waε recovered in the εpleen. Intereεtingly, when the α2,6-εialylated enzyme was treated with acid phosphataεe, the modified enzyme waε redistributed to the liver and spleen, presumably to their reticuloendothelial cells (Fig. 25) . Acid phosphataεe treatment of the non-α2,6-εialylated enzyme reεulted in the recovery of significantly more enzyme than its non-phoεphataεe treated form in the lungε and heart whereaε, leεε waε recovered in the kidney (Fig. 25) . 10. EXAMPLE: OVEREXPRESSION OF HUMAN LYSOSOMAL PROTEINS RESULTS IN THEIR INTRACELLULAR AGGREGATION, CRYSTALLIZATION IN LYSOSOMES, AND SELECTIVE SECRETION The example presented here describeε a method, which iε an inherent feature of the invention and was first noted while overexpressing α-Gal A, whereby certain proteins which are normally intracellularly targeted may be recombinantly expressed in a secreted form. Using several recombinant lysoεomal enzymeε, the reεultε demonstrate that overexpreεεion of εuch proteinε in CHO cellε presumably leads to aggregation which, in turn, causes the protein to be secreted, rather than being targeted to the intracellular veεicleε, in thiε case, lysosomeε, to which they would normally be εent. Thiε εurprising secretion feature facilitateε eaεy purification of the recombinant protein produced. 10.1. MATERIALS AND METHODS 10.1.1. CONSTRUCTION OF PLASMIDS FOR LYSOSOMAL ENZYME OVERPRODUCTION Conεtruction of the α-Gal A expreεεion construct, p91-AGA, has been described supra (Section 6.1.2). The analogouε expreεεion conεtructε for human α-N- acetylgalactoεaminidaεe (deεignated p91-AGB) and acid sphingomyelinaεe (deεignated p91-ASM) and the reεpective tranεient expreεεion of each in COS-l cells have been described (Wang et al., 1990, J. Biol. Chem. 265: 21859-21866; Schuchman et al.. 1991, J. Biol. Chem. 266: 8535-8539) . 10.1.2. CELL CULTURE, ELECTROTRANSFECTION, AND GENE AMPLIFICATION CHO cells were grown and maintained as described supra. in Section 6.1.3. Likewise, electroporation was performed as described supra, in Section 6.1.3. Butyrate stimulation of the α-Gal A expressing CHO cells was performed as previously described (Dorner et al., 1989 J. Biol. Chem. 264:20602-20607) . Briefly, cells were plated in 100 mm diεhes and allowed to grow in 10 ml of DMEM supplemented with 10% dFCS for 2 days. The media was removed and replaced with 10 ml of DMEM supplemented with 10% dFCS containing 5 mM sodium butyrate. Cells were incubated for 16 hr at 37 °C in a C0 2 incubator and then cells and culture media were harvested and the α-Gal A activity was determined. 10.1.3. ULTRASTRUCTURAL AND IMMUNOLABELING STUDIES 0 DG5.3-1000MX cells were grown to confluency in 100 mm disheε. Following trypεinization (0.25% trypεin, EDTA) , they were waεhed twice in phosphate buffered saline (PBS) and pelleted at 1,500 g for 5 min at room temperature. Cells were then fixed for 1 5 hr with 3% glutaraldehyde in PBS, followed by fixation in PBS-buffered 1% Os0 4 for 30 min at room temperature. Sa pleε were then dehydrated with graded εteps of ethanol, infiltrated with propylene oxide and embedded in Embed 812 (Electron Microscopy Sciences, Fort o Washington, PA) . 1 μm sections were cut from representative areas. Ultrathin sections were prepared and stained with uranyl acetate and lead citrate, and then were viewed with an electron microscope (JEM 100 CX, Jeol. USA, Peabody, MA) . 5 For immunodetection, sections were prepared aε above, and after embedding, they were mounted on Formvar-coated nickel gridε (Formvar Scientific, Marietta, OH) , incubated with goat serum in PBS for 30 min at 37°C to block nonspecific binding, washed six 0 timeε with PBS and then incubated with affinity- purified rabbit anti-α-Gal A antibodieε for 1 hr. The εectionε were washed extensively aε above and then were incubated with 10 nm gold particleε conjugated to protein A (Amerεham Corp. , Arlington, IL) for 1 hr at 5 37°C. After waεhing with PBS, εectionε were fixed with 3% glutaraldehyde in PBS for 15 min at room temperature, washed again with PBS and then examined under the electron microscope. 5 10.1.4. SDS-PAGE AND AUTORADIOGRAPHY PAGE gel electrophoreεiε waε carried out under reducing conditionε aε deεcribed by Laemmli (1970 Nature (London) 277:680-685) in 1.5 mm thick slab gels containing 10% acrylamide. The gel was fixed in 10% 0 acetic acid and 20% methanol for 30 min and then soaked in Amplify (Amersham Corp.) for 30 min with agitation. Gels were vacuum dried for 90 min (Hoffer Scientific Instrumentε, San Franciεco, CA) and then autoradiographed with Kodak X Omat AR film (Eastman 5 Kodak Co.) for 4-24 hrs. 10.1.5. IN VITRO STUDIES OF α-GAL A AGGREGATION The possible formation of insoluble α-Gal A aggregates at varying enzyme and hydrogen ion o concentrationε waε inveεtigated. Uεing a stock solution of purified, secreted α-Gal A (16 mg/ml in 10 mM Tris buffer, pH 7.0), appropriate aliquotε were placed in glaεε boroεilicate tubeε and the volumeε were brought to 200 μl with diεtilled water εo that 5 with the addition of 100 μi of the appropriate buffer (0.5 M 2-(N-morpholino)ethanesulfonic acid, at pH 5.0, 5.5, 6.0, 6.5 or 7.0), the final α-Gal A concentrations (0.1-10 mg/ml) would be achieved at specific pH values. After incubation for 10 min at 0 room temperature, the turbidity of each solution waε determined by meaεuring the OD at 650 nm in a spectrophotoroeter (Spectronic 1201, Milton Roy Co. , Rochester, NY) uεing a 1-cm path cuvette. Aε a control, 1 mg/ml of purified, εecreted α-Gal A was 5 mixed with increasing BSA concentrations (0.1-10 mg/ml) and the turbidity of each solution was determined. Similar experiments were performed with solutions of α-Gal A (10 mg/ml) and BSA (2 mg/ml) , at decreasing pH (from pH 7.0 to 5.0) . After incubation and centrifugation as above, the supernatantε and pelletε were εubjected to SDS-PAGE. 10.1.6. ENZYME AND PROTEIN ASSAYS The α-Gal A activitieε in the cell lysates and media were determined uεing 5 mM 4-methylumbelliferyl- α-D-galactopyranoεide (4MU-α-Gal) (Genzyme Corp., Cambridge, MA) aε previously described (Bishop and Desnick, 1981 J. Biol. Chem. 256:1307-1316^ . Briefly, a stock solution of 5 mM 4MU-α-Gal was prepared in 0.1 M citrate/0.2 M phoεphate buffer, pH 4.6, in an ultrasonic bath. The reaction mixture, containing 10 - 50 μl of cell extract and 150 μl of the stock substrate solution, was incubated at 37°C for 10 to 30 min. The reaction was terminated with the addition of 2.3 ml of 0.1 M ethylenediamine. The fluorescence was determined using a Ratio-2 System Fluorometer (Optical Technology Devices, Elmεford, NY) . 1U of activity is the amount of enzyme that hydrolyzed one nmol of subεtrate per hour. The activities of α-mannosidase, 3-galactosidase, 3-hexosaminidase, 3-glucuronidase, acid phosphatase and α-N-acetylgalactosaminidase were measured using the appropriate 4-methylumbelliferyl substrate. The activity of acid sphingomyelinase waε determined according to Gal et al. (1975, N. Eng. J. Med. 293:632-636) . Protein concentrations were quantitated by the fluorescamine method (Bohlen et al.. 1973, Arch. Biochem. Biophys. .155:213-220) as modified by Bishop et al. (1978, Biochem. Biophys. Acta 524:109-120) . 10.2. RESULTS 10.2.1. OVEREXPRESSION RESULTS IN CRYSTALLINE STRUCTURES CONTAINING HUMAN α-GAL A IN MEMBRANE-LIMITED VESICLES Ultrastructural examination of the stably amplified DG5.3-1000MX cellε revealed numerouε 0.25 to 1.5 μm cryεtalline bodies which had ordered triangular lattices in membrane-limited vesicles throughout the cytoplasm (FIG. 26A and 26B) . the repeat within these cryεtalline εtructures was about 20 nm. these structureε were particularly abundant in lysosomes (FIG. 28A) and in vesicles which appeared to be dilated TGN (FIG. 28B) (Hand and Oliver, 1984, J. Histochem. Cytochem. .42 . : 403-442; Griffith and Simonε, 1986, Science 234:438-443; McCracken, 1991, in Intracellular Traficking of Proteinε, Steer and Hanover, edε., Cambridge Univerεity Preεε, New York pp. 461-485) . Of note, normal Golgi εtructureε were not obεerved in theεe cellε, whereaε Golgi complexes were readily identified in the parental DG44 cells (FIG. 26E, inset) . When osmium-glutaraldehyde fixed εectionε of the DG5.3-1000MX cellε were incubated with affinity-purified rabbit anti-human α-Gal A antibodies and then with protein A-conjugated gold, these crystalline structures were specifically stained by gold particles (FIG. 26C and 26D) . That the crystalline structures were imraunogold labeled, even though the sectionε were fixed in oεmium- glutaraldehyde, suggested that these structures were primarily, if not solely, composed of the human enzyme. To determine whether the crystalline εtructureε were preεent in clones expressing lower levels of α- Gal A, clones DG5.3-0MX, -1.3Mx, -250Mx, and -lOOOMx were grown to confluency and examined by electron microscopy. Although the TGN was increasingly dilated with increasing α-Gal A expression, only the DG5.3- lOOOMx clone contained crystalline arrays in 5 lysosomes. Similarly, clone DG5.3-l.3Mx was stimulated with 5mM sodium butyrate for 18 hrs to increase transcription of the integrated vector containing the α-Gal A cDNA and then examined ultrastructurally. Compared to the untreated clone, 0 butyrate treatment reεulted in the preεence of dilated organelles including many membrane-bound structureε containing denεe material. Theεe reεultε indicated that crystal formation was α-Gal A concentration dependent. Furthermore, to asseεs whether crystal 5 formation was specific to α-Gal A, clone AGB14.8- lOOOMx, which overexpresseε α-N- Acetylgalactoεa inidaεe, waε examined. No cryεtalline arrayε were observed, but numerous dilated structureε were seen similar to those in the DG5.3-l.3Mx clone, o suggesting that expresεion of the recombinant α-N- acetylgalactoεaminidaεe had not reached the critical level neceεεary for cryεtal formation. 10.2.2. α-GAL A AND α-N-ACETYLGALACTOSAMINIDASE AGGREGATE AT HIGH CONCENTRATION AND LOW pH 5 Since it was conceivable that the overexpression of α-Gal A reεulted in the formation of εoluble and particulate aggregateε that did not bind to or were inefficiently bound by the M6PR and/or the εulfotrans- feraεe in the TGN, the poεεible aggregation of α-Gal A 0 waε aεεeεsed in vitro at varying enzyme and hydrogen ion concentrations. As shown in FIG. 27A, the amount of α-Gal A precipitated, compared to about 30% (>2 x 10 6 U) at pH 5.0. At pH 6.0, the estimated pH of the TGN (Griffith and Simons, 1986, Science 234 : 438-443), 5 about 12% of the enzyme formed particulate aggregates that could be pelleted by centrifugation at 15,υυ0 x g. FIG. 27B shows that the turbidity, as a measure of aggregation (Halper and Stere, 1977, Arch. Biochem. Biophys. 184 : 529-534) , of εolutions containing 0.1 to 10 mg/ml of α-Gal A at either pH 5.0 or 7.0 increased as a function of enzyme concentration. Moreover, the turbidity of a 1 mg/ml α-Gal A solution was esεentially unaffected by the preεence of increasing albumin concentrations from 0.1 to 10 mg/ml at pH 5.0 (FIG. 27B; control). Finally, electrophoresis of the supernatant and pellet fractions from solutionε containing α-Gal A (10 mg/ml) and bovine serum albumin (BSA) (2 mg/ml) incubated at varying hydrogen ion concentrations revealed that the increasing precipitation of α-Gal A with decreasing pH was enzyme specific, as the BSA did not precipitate over this pH range (FIG. 27C) . 10.3. DISCUSSION An "aggregation-secretion" model is proposed to account for the rerouting of human α-Gal A as a prototype for overproduced targeted proteins. As depicted in FIG. 28, the overproduced enzyme is normally synthesized and procesεed until it reacheε the trans-Golgi network (TGN) . In this structure, the overproduced enzyme is accumulated and subjected to a markedly more acidic environment, (app. pH 6.0), which leads to increased protein-protein interactions that generate soluble and particulate α-Gal A aggregates. As a result of such aggregation, the enzyme's M6P moieties become inaccesεible or leεε accessible for binding to the MGPR. The aggregates with inaccessible MGP moieties, by default, are rerouted via the conεtitutive secretory pathway (Helms et al.. 1990, J. Biol. Chem. 265: 20,027-20,032). The cellular response, therefore, to the overproduction of lysosome-targeted protein is to transport those proteins having available MGP residues to the lysosome and to reroute the majority of the overproduced, and presumably aggregated, proteins through the constitutive secretion pathway. The fact that large amounts of recombinant human α-Gal A are secreted by CHO cells permits the scaled-up production and easy purification of the recombinant enzyme for crystallography and for trials of enzyme replacement therapy in patients with Fabry disease. In addition, it is clear that the amplification εerieε of overexpressing α-Gal A CHO cellε provideε a unique experimental mammalian εyεtem to efficiently characterize the bioεynthesis, poεttranslational modifications, and mechanisms responεible for the lyεoεomal targeting and εelective secretion of this prototype lysoεomal enzyme, thereby providing further insight into the nature of protein transport and sorting in mammalian cellε. Thuε, the overexpreεεion of lysosomal and perhaps other targeted proteins in CHO cells provides a convenient method for producing large amounts of the protein for structural analyseε and/or therapeutic applicationε, aε well aε providing a uεeful approach to εtudy protein bioεynthesiε and sorting. 11. DEPOSIT OF MICROORGANISMS The following Εy_ coli strains carrying the listed plasmidε have been deposited with the Agricultural Research Culture Collection (NRRL) , Peoria, IL and have been assigned the following accesεion number: Host Cell Strain Plasmid Accession No. E. coli kl2 p91.AGA B 18722 E. Coli kl2 pAGA-PA B 18723 The present invention is not to be limited in scope by the microorganisms deposited since the deposited embodimentε are intended aε illuεtration of individual aspects of the invention and any microorganisms, or constructs which are functionally equivalent are within the εcope of this invention. Indeed various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Desnick, Robert J. Bishop, David F. Ioannou, Yianniβ A. (ii) TITLE OF INVENTION: Cloning and Expression of Biologically Active alpha-Galactosidaβe A (iii) NUMBER OF SEQUENCES: 13 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: PENNIE & EDMONDS (B) STREET: 1155 Avenue of the Americas (C) CITY: New ϊork (D) STATE: New York (E) COUNTRY: U.S.A. (F) ZIP: 10036 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version 1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: US 07/983,451 (B) FILING DATE: 30-NOV-1992 (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Coruzzi, Laura A. (B) REGISTRATION NUMBER: 07/983,451 (C) REFERENCE/DOCKET NUMBER: 6923-030 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 212-790-9090 (B) TELEFAX: 212-869-8864/9741 (C) TELEX: 66141 PENNIE (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1393 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 61..1350 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: AGGTTAATCT TAAAAGCCCA GGTTACCCGC GGAAATTTAT GCTGTCCGGT CACCGTGACA 60 ATG CAG CTG AGG AAC CCA GAA CTA CAT CTG GGC TGC GCG CTT GCG CTT 108 Met Gin Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala Leu 1 5 10 15 CGC TTC CTG GCC CTC GTT TCC TGG GAC ATC CCT GGG GCT AGA GCA CTG 156 Arg Phe Leu Ala Leu Val Ser Trp Asp lie Pro Gly Ala Arg Ala Leu 20 25 30 GAC AAT GGA TTG GCA AGG ACG CCT ACC ATG GGC TGG CTG CAC TGG GAG 204 Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp Glu 35 40 45 CGC TTC ATG TGC AAC CTT GAC TGC CAG GAA GAG CCA GAT TCC TGC ATC 252 Arg Phe Met Cys Asn Leu Asp Cys Gin Glu Glu Pro Asp Ser Cys lie 50 55 60 AGT GAG AAG CTC TTC ATG GAG ATG GCA GAG CTC ATG GTC TCA GAA GGC 300 Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu Gly 65 70 75 80 TGG AAG GAT GCA GGT TAT GAG TAC CTC TGC ATT GAT GAC TGT TGG ATG 348 Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys lie Asp Asp Cys Trp Met 85 90 95 GCT CCC CAA AGA GAT TCA GAA GGC AGA CTT CAG GCA GAC CCT CAG CGC 396 Ala Pro Gin Arg Asp Ser Glu Gly Arg Leu Gin Ala Asp Pro Gin Arg 100 105 110 TTT CCT CAT GGG ATT CGC CAG CTA GCT AAT TAT GTT CAC AGC AAA GGA 444 Phe Pro His Gly lie Arg Gin Leu Ala Asn Tyr Val His Ser Lys Gly 115 120 125 CTG AAG CTA GGG ATT TAT GCA GAT GTT GGA AAT AAA ACC TGC GCA GGC 492 Leu Lys Leu Gly lie Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala Gly 130 135 140 TTC CCT GGG AGT TTT GGA TAC TAC GAC ATT GAT GCC CAG ACC TTT GCT 540 Phe Pro Gly Ser Phe Gly Tyr Tyr Asp lie Asp Ala Gin Thr Phe Ala 145 150 155 160 GAC TGG GGA GTA GAT CTG CTA AAA TTT GAT GGT TGT TAC TGT GAC AGT 588 Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser 165 170 175 TTG GAA AAT TTG GCA GAT GGT TAT AAG CAC ATG TCC TTG GCC CTG AAT 636 Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu Asn 180 185 190 AGG ACT GGC AGA AGC ATT GTG TAC TCC TGT GAG TGG CCT CTT TAT ATG 684 Arg Thr Gly Arg Ser lie Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met 195 200 205 TGG CCC TTT CAA AAG CCC AAT TAT ACA GAA ATC CGA CAG TAC TGC AAT 732 Trp Pro Phe Gin Lys Pro Asn Tyr Thr Glu lie Arg Gin Tyr Cys Asn 210 215 220 CAC TGG CGA AAT TTT GCT GAC ATT GAT GAT TCC TGG AAA AGT ATA AAG 780 His Trp Arg Asn Phe Ala Asp lie Asp Asp Ser Trp Lys Ser lie Lys 225 230 235 240 AGT ATC TTG GAC TGG ACA TCT TTT AAC CAG GAG AGA ATT GTT GAT GTT 828 Ser lie Leu Asp Trp Thr Ser Phe Asn Gin Glu Arg lie Val Asp Val 245 250 255 GCT GGA CCA GGG GGT TGG AAT GAC CCA GAT ATG TTA GTG ATT GGC AAC 876 Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val lie Gly Asn 260 265 270 TTT GGC CTC AGC TGG AAT CAG CAA GTA ACT CAG ATG GCC CTC TGG GCT 924 Phe Gly Leu Ser Trp Asn Gin Gin Val Thr Gin Met Ala Leu Trp Ala 275 280 285 ATC ATG GCT GCT CCT TTA TTC ATG TCT AAT GAC CTC CGA CAC ATC AGC 972 lie Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His lie Ser 290 295 300 CCT CAA GCC AAA GCT CTC CTT CAG GAT AAG GAC GTA ATT GCC ATC AAT 1020 Pro Gin Ala Lys Ala Leu Leu Gin Asp Lys Asp Val lie Ala lie Asn 305 310 315 320 CAG GAC CCC TTG GGC AAG CAA GGG TAC CAG CTT AGA CAG GGA GAC AAC 1068 Gin Asp Pro Leu Gly Lys Gin Gly Tyr Gin Leu Arg Gin Gly Asp Asn 325 330 335 TTT GAA GTG TGG GAA CGA CCT CTC TCA GGC TTA GCC TGG GCT GTA GCT 1116 Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val Ala 340 345 350 ATG ATA AAC CGG CAG GAG ATT GGT GGA CCT CGC TCT TAT ACC ATC GCA 1164 Met lie Asn Arg Gin Glu lie Gly Gly Pro Arg Ser Tyr Thr lie Ala 355 360 365 GTT GCT TCC CTG GGT AAA GGA GTG GCC TGT AAT CCT GCC TGC TTC ATC 1212 Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe lie 370 375 380 ACA CAG CTC CTC CCT GTG AAA AGG AAG CTA GGG TTC TAT GAA TGG ACT 1260 Thr Gin Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp Thr 385 390 395 400 TCA AGG TTA AGA AGT CAC ATA AAT CCC ACA GGC ACT GTT TTG CTT CAG 1308 Ser Arg Leu Arg Ser His lie Asn Pro Thr Gly Thr Val Leu Leu Gin 405 410 415 CTA GAA AAT ACA ATG CAG ATG TCA TTA AAA GAC TTA CTT TAAAAAAAAA 1357 Leu Glu Asn Thr Met Gin Met Ser Leu Lys Asp Leu Leu 420 425 430 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAA 1393 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 429 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Gin Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala Leu 1 5 10 15 Arg Phe Leu Ala Leu Val Ser Trp Asp lie Pro Gly Ala Arg Ala Leu 20 . 25 30 Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp Glu 35 40 45 Arg Phe Met Cys Asn Leu Asp Cys Gin Glu Glu Pro Asp Ser Cys lie 50 55 60 Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu Gly 65 70 75 80 Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys lie Asp Asp Cys Trp Met 85 90 95 Ala Pro Gin Arg Asp Ser Glu Gly Arg Leu Gin Ala Asp Pro Gin Arg 100 105 110 Phe Pro His Gly lie Arg Gin Leu Ala Asn Tyr Val Hie Ser Lys Gly 115 120 125 Leu Lys Leu Gly lie Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala Gly 130 135 140 Phe Pro Gly Ser Phe Gly Tyr Tyr Asp lie Asp Ala Gin Thr Phe Ala 145 150 155 160 Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser 165 170 175 Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu Asn 180 185 190 Arg Thr Gly Arg Ser He Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met 195 200 205 Trp Pro Phe Gin Lys Pro Asn Tyr Thr Glu He Arg Gin Tyr Cys Asn 210 215 220 His Trp Arg Asn Phe Ala Asp He Asp Asp Ser Trp Lys Ser He Lyε 225 230 235 240 Ser He Leu Asp Trp Thr Ser Phe Asn Gin Glu Arg He Val Asp Val 245 250 255 Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val He Gly Asn 260 265 270 Phe Gly Leu Ser Trp Asn Gin Gin Val Thr Gin Met Ala Leu Trp Ala 275 280 285 He Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg Hiε He Ser 290 295 300 Pro Gin Ala Lys Ala Leu Leu Gin Asp Lys Asp Val He Ala He Asn 305 310 315 320 Gin Asp Pro Leu Gly Lys Gin Gly Tyr Gin Leu Arg Gin Gly Asp Asn 325 330 335 Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val Ala 340 345 350 Met He Asn Arg Gin Glu He Gly Gly Pro Arg Ser Tyr Thr He Ala 355 360 365 Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe He 370 375 380 Thr Gin Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp Thr 385 390 395 400 Ser Arg Leu Arg Ser His He Asn Pro Thr Gly Thr Val Leu Leu Glr. 405 410 415 Leu Glu Asn Thr Met Gin Met Ser Leu Lys Asp Leu Leu 420 425 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 411 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Met Leu Leu Lys Thr Val Leu Leu Leu Gly His Val Ala Gin Val Leu 1 5 10 15 Met Leu Asp Asn Gly Leu Leu Gin Thr Pro Pro Met Gly Trp Leu Ala 20 25 30 Trp Glu Arg Phe Arg Cys Asn He Asn Cys Asp Glu Asp Pro Lys Asn 35 40 45 Cys He Ser Glu Gin Leu Phe Met Glu Met Ala Asp Arg Met Ala Gin 50 55 60 Asp Gly Trp Arg Asp Met Gly Tyr Thr Tyr Leu Asn He Asp Asp Cys 65 70 75 80 Trp He Gly Gly Arg Asp Ala Ser Gly Arg Leu Met Pro Asp Pro Lys 85 90 95 Arg Phe Pro His Gly He Pro Phe Leu Ala Asp Tyr Val His Ser Leu 100 105 110 Gly Leu Lys Leu Gly He Tyr Ala Asp Met Gly Asn Phe Thr Cys Met 115 120 125 Gly Tyr Pro Gly Thr Thr Leu Asp Lys Val Val Gin Asp Ala Gin Thr 130 135 140 Phe Ala Glu Trp Lys Val Asp Met Leu Lys Leu Asp Gly Cys Phe Ser 145 150 155 160 Thr Pro Glu Glu Arg Ala Gin Gly Tyr Pro Lys Met Ala Ala Ala Leu 165 170 175 Asn Ala Thr Gly Arg Pro He Ala Phe Ser Cys Ser Trp Pro Ala Tyr 180 185 190 Glu Gly Gly Leu Pro Pro Arg Val Asn Tyr Ser Leu Leu Ala Asp He 195 200 205 Cys Asn Leu Trp Arg Asn Tyr Asp Asp He Gin Asp Ser Trp Trp Ser 210 215 220 Val Leu Ser He Leu Asn Trp Phe Val Glu His Gin Asp He Leu Gin 225 230 235 240 Pro Val Ala Gly Pro Gly His Trp Asn Asp Pro Asp Met Leu Leu He 245 250 255 Gly Asn Phe Gly Leu Ser Leu Glu Gin Arg Ser Arg Ala Gin Met Ala 260 265 270 Leu Trp Thr Val Leu Ala Ala Pro Leu Leu Met Ser Thr Asp Leu Arg 275 280 285 Thr He Ser Ala Gin Asn Met Asp He Leu Gin Asn Pro Leu Met He 290 295 300 Lys He Asn Gin Asp Pro Leu Gly He Gin Gly Arg He His Lys Glu 305 310 315 320 Lys Ser Leu He Glu Val Tyr Met Arg Pro Leu Ser Asn Lys Ala Ser 325 330 335 Ala Leu Val Phe Phe Ser Cys Arg Thr Asp Met Pro Tyr Arg Tyr His 340 345 350 Ser Ser Leu Gly Gin Leu Asn Phe Thr Gly Ser Val He Tyr Glu Ala 355 360 365 Gin Asp Val Tyr Ser Gly Asp He He Ser Gly Leu Arg Asp Glu Thr 370 375 380 Asn Phe Thr Val He He Asn Pro Ser Gly Val Val Met Trp Tyr Leu 385 390 395 400 Tyr Pro He Lys Asn Leu Glu Met Ser Gin Gin 405 410 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 404 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met Phe Ala Phe Tyr Phe Leu Thr Ala Cys He Ser Leu Lys Gly Val 1 5 10 15 Phe Gly Ser Tyr Asn Gly Leu Gly Leu Thr Pro Gin Met Gly Trp Asp 20 25 30 Asn Trp Asn Thr Phe Ala Cys Asp Val Ser Glu Gin Leu Leu Leu Asp 35 40 45 Thr Ala Asp Arg He Ser Asp Leu Gly Leu Lys Asp Met Gly Tyr Lys 50 55 60 Tyr He He Leu Asp Asp Cys Trp Ser Ser Gly Arg Asp Ser Asp Gly 65 70 75 80 Phe Leu Val Ala Asp Glu Gin Lys Phe Pro Asn Gly Met Gly His Val 85 90 95 Ala Asp His Leu His Asn Asn Ser Phe Leu Phe Gly Met Tyr Ser Ser 100 105 110 Ala Gly Glu Tyr Thr Cys Ala Gly Tyr Pro Gly Ser Leu Gly Arg Glu 115 120 125 Glu Glu Asp Ala Gin Phe Phe Ala Asn Asn Arg Val Asp Tyr Leu Lys 130 135 140 Tyr Asp Asn Cys Tyr Asn Lys Gly Gin Phe Gly Thr Pro Glu Ser Tyr 145 150 155 160 Arg Lys Met Ser Asp Ala Leu Asn Lys Thr Gly Arg Pro He Phe Tyr 165 170 175 Ser Cys Asn Trp Gly Leu Tyr Gly Ser Gly He Ala Asn Ser Trp Arg 180 185 190 Met Ser Gly Asp Val Thr Ala Glu Phe Thr Arg Pro Asp Ser Cys Pro 195 200 205 Asp Gly Tyr Tyr Ala Gly Phe Ser He Met Asn He Leu Asn Lys Ala 210 215 220 Ala Pro Met Gly Gin Asn Ala Gly Val Gly Gly Trp Asn Asp Leu Asp 225 230 235 240 Asn Leu Glu Val Gly Val Gly Asn Leu Thr Asp Asp Glu Glu Lys Ala 245 250 255 His Phe Ser Met Trp Ala Met Val Lys Ser Pro Leu He He Gly Ala 260 265 270 Asn Val Asn Asn Leu Lys Ala Ser Ser Tyr Ser He Tyr Ser Gin Ala 275 280 285 Ser Val He Ala He Asn Gin Asp Ser Asn Gly He Pro Ala Arg Val 290 295 300 Ser Asp Thr Asp Glu Tyr Gly Glu He Trp Ser Gly Pro Leu Asp Asn 305 310 315 320 Gly Asp Gin Val Val Ala Leu Leu Asn Gly Gly Ser Val Ser Arg Pro 325 330 335 Met Asn Thr Thr Leu Glu He Asp Ser Leu Gly Lys Lys Leu Thr Ser 340 345 350 Thr Asp Asp Leu Trp Ala Asn Arg Val Thr Ala Ser He Gly Arg Lys 355 360 365 Thr Gly Leu Tyr Glu Tyr Lys Asp Gly Leu Lys Asn Arg Leu Gly Gin 370 375 380 Lys Gly Ser Leu He Leu Asn Val Pro Ala His He Ala Phe Arg Leu 385 390 395 400 Arg Pro Ser Ser (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: Glu Gin Thr He Ala Asp Thr Leu Gly Pro Gly Gly 1 5 10 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Pro Ser Val He Tyr Gly Asn Val Arg Asn 1 5 10 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Glu Val Ala Cys Leu Val Asp Ala Asn Gly He Gin Pro 1 5 10 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 297 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..279 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GAA TGG ACT TCA AGG TTA AGA AGT CAC ATA AAT CCC ACA GGA ACT GTT 48 Glu Trp Thr Ser Arg Leu Arg Ser His He Asn Pro Thr Gly Thr Val 1 5 10 15 TTG CTT CAG CTA GAA AAT ACA ATG CAG ATG TCA TTA AAA GAC TTA CTT 96 Leu Leu Gin Leu Glu Asn Thr Met Gin Met Ser Leu Lys Asp Leu Leu 20 25 30 CCG GCT GGT CCG GCG CAA CAC GAT GAA GCT CAA CAA AAT GCT TTT TAT 3,44 Pro Ala Gly Pro Ala Gin His Asp Glu Ala Gin Gin Asn Ala Phe Tyr 35 40 45 CAA GTC TTA AAT ATG CCT AAC TTA AAT GCT GAT CAA CGC AAT GGT TTT 192 Gin Val Leu Asn Met Pro Asn Leu Asn Ala Asp Gin Arg Asn Gly Phe 50 55 60 ATC CAA AGC CTT AAA GAT GAT CCA AGC CAA AGT GCT AAC GTT TTA GGT 240 He Gin Ser Leu Lys Asp Asp Pro Ser Gin Ser Ala Asn Val Leu Gly 65 70 75 80 GAA GCT CAA AAA CTT AAT GAC TCT CAA GCT CCA AAA TAAGGATCCC 286 Glu Ala Gin Lys Leu Asn Asp Ser Gin Ala Pro Lys 85 90 GGAATTCGGC C 297 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 92 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Glu Trp Thr Ser Arg Leu Arg Ser His He Asn Pro Thr Gly Thr Val 1 5 10 15 Leu Leu Gin Leu Glu Asn Thr Met Gin Met Ser Leu Lys Asp Leu Leu 20 25 30 Pro Ala Gly Pro Ala Gin His Asp Glu Ala Gin Gin Asn Ala Phe Tyr 35 40 45 Gin Val Leu Asn Met Pro Asn Leu Asn Ala Asp Gin Arg Asn Gly Phe 50 55 60 He Gin Ser Leu Lys Asp Asp Pro Ser Gin Ser Ala Asn Val Leu Gly 65 70 75 80 Glu Ala Gin Lys Leu Asn Asp Ser Gin Ala Pro Lys 85 90 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: CCGAATTCAT GCTGTCCGGT CACCGTG 27 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: CGCCGGACCA GCCGGAAGTA AGTCTTTTAA TG 32 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: CCGGCTGGTC CGGCGCAACA CGATGAAGCT 30 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GGCCGAATTC CGGGATCCTT ATTTTGGAGC TTGAGA 36 lnternational Apphcation No- PCT/ / MICROORGANISMS Optional Sheet in connection with the microorganism referred to on page 98, lines 29-33 of the description A. IDENTIFICATION OF DEPOSIT ' Further deposits are identified on an additional sheet Name of depositary institution ' Agricultural Research Culture Collection (NRRL) International Depository Authority Address of depositary institution (including postal code and country) 1815 N. University Street Peoria, IL 61604 US Date of deposit ' October 5, 1990 Accession Number * B 18722 B. ADDITIONAL INDICATIONS ' (leaw blank if not applicable) Thu information u continued on • attached »h__ct C. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE ' «*.. D. SEPARATE FURNISHING OF INDICATIONS ' (ic« b n _r no. a lic ble) The indicationa listed below will be submitted to the International Bureau later ' (Specify the general nature or the indications e g "Accession Number of Oepoait") E -... 0 h TT1his sheet was received with D The date of receipt (from the applicant) by the International Bureau " (Authorized Officer) Form PCT/RO/134 (January 1981 ) International Application No: PCT/ Form PCT/RO/134 (cont.) Agricultural Research Culture Collection (NRRL) International Depository Authority 1815 N. University Street Peoria. IL 61604 US Accession No. Date of Deposit B 18723 October 5, 1990

Claims: WHAT IS CLAIMED IS: 1. A method for producing human α-galactosidase A comprising: (a) culturing a transformed mammalian cell transformed with a β galactoside 2,6- εialyltransferase gene which expresses β- galactoside α2, 6-sialyltransferase such that the cell is capable of peptide sialylation, further containing a heterologous chromosomally integrated nucleotide sequence encoding human α-galactosidase A operatively associated with a nucleotide εequence that regulates gene expression and a selectable marker controlled by the same or a different regulatory sequence, so that the α- galactosidase A nucleotide sequence is stably overexpressed and an enzymatically active, sialylated glycoform of the α- galactosidase A enzyme is secreted by the mammalian cell; and (b) isolating enzymatically active α- galactosidase A enzyme from the mammalian cell culture. 2. The method according to Claim 1 in which the nucleotide sequence encoding α-galactosidase A comprises the sequence depicted in FIG. 1A-1C from nucleotide number 1 to 1299. 3. The method according to Claim 1 in which the nucleotide sequence encoding α-galactosidase A comprises the sequence depicted in FIG. 1A-1C from nucleotide number 91 to 1299. 4. The method according to Claim 1 in which the nucleotide sequence that regulates gene expression comprises a viral promoter. 5. The method according to Claim 1 in which the nucleotide sequence that regulates gene expression comprises an inducible promoter. 6. The method according to Claim 1 wherein, in the presence of selection, the chromosomally integrated nucleotide sequences are amplified. 7. The method according to Claim 1 in which the selectable marker is dihydrofolate reductase. 8. The method according to claim 6 in which the selectable marker is dihydrofolate reductase and the selection is methotrexate. 9. The method according to Claim 1 in which the mammalian cell is a Chinese hamster ovary cell line. 10. A method for producing secreted proteins, comprising: (a) culturing a transformed mammalian cell containing a nucleotide sequence encoding the secreted protein operatively associated with a nucleotide sequence that regulates gene expression so that the nucleotide sequence is stably expressed and the protein is stably expressed and the protein iε stably produced by the transformed mammalian cell forming aggregates which are secreted into the mammalian cell culture; and (b) isolating said protein from the mammalian cell culture. 11. The method according to Claim 10 in which the protein is an endoplasmic reticulum-targeted, a Golgi-targeted, or a membrane-targeted protein. 12. The method according to Claim 10 in which "the protein is a lysosomal protein. 13. The method according to Claim 10 in which the protein is biologically active α-galactosidase A. 14. The method according to Claim 10 in which the protein is biologically active α-N- acetylgalactosaminidase. 15. The method according to Claim 10 in which the protein is acid εphingomyelinaεe.

Inventors: BISHOP DAVID F, DESNICK ROBERT J, IOANNOU YIANNIS A, BISHOP, DAVID, F., DESNICK, ROBERT, J., IOANNOU, YIANNIS, A.
Applicants: SINAI SCHOOL MEDICINE, THE MOUNT SINAI SCHOOL OF MEDICINE OF THE CITY UNI
Application year: 1993
Main classification: C12N15
IPC codes: A61K38, C07K14, C12N9, C12N15, C12R1