Expression, purification and characterization of recombinant human N-acetylgalactosamine-6-sulphatase.
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.
Bielicki
J
J
Department of Chemical Pathology, Women's and Children's Hospital, North Adelaide, South Australia.
Fuller
M
M
Guo
X H
XH
Morris
C P
CP
Hopewood
J J
JJ
Anson
D S
DS
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
D017077:Culture Media, Conditioned; D018076:DNA, Complementary; D011994:Recombinant Proteins; D002811:Chondroitinsulfatases