title: Expression, purification and characterization of recombinant human N-acetylgalactosamine-6-sulphatase.
abstract: Full-length cDNA sequences encoding human N-acetylgalactosamine-6-sulphatase were stably expressed in Chinese hamster ovary cells under the transcriptional control of the human polypeptide chain elongation factor 1 alpha gene promoter. A clonal cell line overexpressing recombinant N-acetylgalactosamine-6-sulphatase to a level of approx. 3 mg/l of culture medium was isolated. The secreted precursor enzyme was purified to homogeneity by a two-column procedure with an overall yield of 53% of the activity. The physical and catalytic parameters of the recombinant enzyme were similar to those of the mature form isolated from liver. On SDS/PAGE and gel filtration, recombinant N-acetylgalactosamine-6-sulphatase had a native molecular mass of 58-60 kDa. Recombinant N-acetylgalactosamine-6-sulphatase was endocytosed by mucopolysaccharidosis IVA fibroblasts via the mannose-6-phosphate receptor-mediated pathway and was efficiently localized to lysosomes.
authors.affiliation: Department of Chemical Pathology, Women's and Children's Hospital, North Adelaide, South Australia.
authors.forename: J
authors.initials: J
authors.lastname: Bielicki
authors.forename: M
authors.initials: M
authors.lastname: Fuller
authors.forename: X H
authors.initials: XH
authors.lastname: Guo
authors.forename: C P
authors.initials: CP
authors.lastname: Morris
authors.forename: J J
authors.initials: JJ
authors.lastname: Hopewood
authors.forename: D S
authors.initials: DS
authors.lastname: Anson
mesh_terms: D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002811:Chondroitinsulfatases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006224:Cricetinae; D017077:Culture Media, Conditioned; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004705:Endocytosis; D005347:Fibroblasts / Q000201:enzymology; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009085:Mucopolysaccharidosis IV / Q000201:enzymology; D011994:Recombinant Proteins / Q000302:isolation & purification
chemical_list: D017077:Culture Media, Conditioned; D018076:DNA, Complementary; D011994:Recombinant Proteins; D002811:Chondroitinsulfatases