Expression, purification and characterization of recombinant human N-acetylgalactosamine-6-sulphatase

J. Bielicki; M. Fuller; X. H. Guo; C. P. Morris; J. J. Hopwood; D. S. Anson

doi:10.1042/bj3110333

Expression, purification and characterization of recombinant human N-acetylgalactosamine-6-sulphatase

J. Bielicki, M. Fuller, X. H. Guo, C. P. Morris, J. J. Hopwood, D. S. Anson

Hopwood Centre For Neurobiology

Research output: Contribution to journal › Article › peer-review

33 Citations (Scopus)

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 1α gene promoter. A clonal cell line overexpressing recombinant N-acetylgalactosamine-6-sulphatase to a level of approx. 3 mg/1 of culture medium was isolated. The secreted precursor enzyme was purifed 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-amine-6-sulphatase was endocytosed by mucopolysaccharidosis IVA fibroblasts via the mannose-6-phosphate receptor-mediated pathway and was efficiently localized to lysosomes.

Original language	English
Pages (from-to)	333-339
Number of pages	7
Journal	Biochemical Journal
Volume	311
Issue number	1
DOIs	https://doi.org/10.1042/bj3110333
Publication status	Published or Issued - 1995

ASJC Scopus subject areas

Biochemistry
Molecular Biology
Cell Biology

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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 1α gene promoter. A clonal cell line overexpressing recombinant N-acetylgalactosamine-6-sulphatase to a level of approx. 3 mg/1 of culture medium was isolated. The secreted precursor enzyme was purifed 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-amine-6-sulphatase was endocytosed by mucopolysaccharidosis IVA fibroblasts via the mannose-6-phosphate receptor-mediated pathway and was efficiently localized to lysosomes.",

author = "J. Bielicki and M. Fuller and Guo, {X. H.} and Morris, {C. P.} and Hopwood, {J. J.} and Anson, {D. S.}",

year = "1995",

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T1 - Expression, purification and characterization of recombinant human N-acetylgalactosamine-6-sulphatase

AU - Bielicki, J.

AU - Fuller, M.

AU - Guo, X. H.

AU - Morris, C. P.

AU - Hopwood, J. J.

AU - Anson, D. S.

PY - 1995

Y1 - 1995

N2 - 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 1α gene promoter. A clonal cell line overexpressing recombinant N-acetylgalactosamine-6-sulphatase to a level of approx. 3 mg/1 of culture medium was isolated. The secreted precursor enzyme was purifed 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-amine-6-sulphatase was endocytosed by mucopolysaccharidosis IVA fibroblasts via the mannose-6-phosphate receptor-mediated pathway and was efficiently localized to lysosomes.

AB - 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 1α gene promoter. A clonal cell line overexpressing recombinant N-acetylgalactosamine-6-sulphatase to a level of approx. 3 mg/1 of culture medium was isolated. The secreted precursor enzyme was purifed 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-amine-6-sulphatase was endocytosed by mucopolysaccharidosis IVA fibroblasts via the mannose-6-phosphate receptor-mediated pathway and was efficiently localized to lysosomes.

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JO - Biochemical Journal

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Expression, purification and characterization of recombinant human N-acetylgalactosamine-6-sulphatase

Abstract

ASJC Scopus subject areas

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