Please use this identifier to cite or link to this item: https://hdl.handle.net/2440/7014
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Type: Journal article
Title: Recombinant human sulphamidase: expression, amplification, purification and characterization
Author: Bielicki, J.
Hopwood, J.
Melville, E.
Anson, D.
Citation: Biochemical Journal, 1998; 329(1):145-150
Publisher: PORTLAND PRESS
Issue Date: 1998
ISSN: 0264-6021
1470-8728
Abstract: Mucopolysaccharidosis type IIIA (MPS IIIA, Sanfilippo A syndrome) is a lysosomal storage disease that causes a profound neurological deterioration. The disorder is caused by a deficiency of the lysosomal enzyme sulphamidase which is a requisite for the degradation of heparan sulphate. To facilitate the development of enzyme-replacement strategies for MPS IIIA patients, we have constructed a high-level expression system for recombinant human sulphamidase in Chinese hamster ovary (CHO) cells. An expression construct containing a methotrexate-resistant dihydrofolate reductase (DHFR) gene allowed amplification of expression levels from less than 1 mg of sulphamidase per litre of culture medium to approx. 15 mg/l. Unlike many cell lines made by gene amplification in DHFR-deficient CHO cells, and utilizing the normal DHFR gene, these cell lines appeared to be stable in the absence of selective pressure. Recombinant human sulphamidase was purified from unamplified and amplified cell lines. The native enzyme was found to be a dimer of 115 kDa. Denaturing and reducing SDS/PAGE revealed a subunit size of 62 kDa. Kinetic analysis demonstrated that the recombinant enzyme had broadly similar kinetic characteristics to sulphamidase purified from liver. Recombinant human sulphamidase was able to correct the storage phenotype of MPS IIIA fibroblasts after endocytosis via the mannose-6-phosphate receptor.
Keywords: Cell Line
CHO Cells
Fibroblasts
Animals
Humans
Mucopolysaccharidosis III
Hydrolases
Recombinant Proteins
Genetic Markers
Chromatography, Ion Exchange
Electrophoresis, Polyacrylamide Gel
Gene Expression
Protein Conformation
Dimerization
Kinetics
Genetic Vectors
Cricetinae
DOI: 10.1042/bj3290145
Appears in Collections:Aurora harvest
Paediatrics publications

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