Please use this identifier to cite or link to this item:
Scopus Web of ScienceĀ® Altmetric
Type: Journal article
Title: Expression, Purification and Characterization of Recombinant Caprine N-Acetylglucosamine-6-Sulphatase
Author: Litjens, T.
Bielicki, J.
Anson, D.
Friderici, K.
Jones, M.
Hopwood, J.
Citation: Biochemical Journal, 1997; 327(1):89-94
Issue Date: 1997
ISSN: 0264-6021
Abstract: Mucopolysaccharidosis type IIID or Sanfilippo D syndrome is a lysosomal storage disorder caused by the deficiency of N-acetylglucosamine-6-sulphatase (Glc6S). In addition to human patients, a Nubian goat with this disorder has been described and the caprine Glc6S (cGlc6S) cDNA cloned. In this study, the full-length cGlc6S cDNA was inserted into the expression vector, pEFNeo, which placed the cGlc6S cDNA under the transcriptional control of the human polypeptide chain elongation factor promoter. The pEFNeo expression vector also contains the human growth hormone polyadenylation signal and the genes encoding resistance to ampicillin and G418. The cGlc6S expression construct was electroporated into Chinese hamster ovary (CHO-K1) cells, and stably transfected clones were isolated. One clone, CHOrcGlc6S.17, which secreted the highest Glc6S activity into the culture medium, was selected and cultured in cell factories. The secreted recombinant cGlc6S (rcGlc6S) precursor was purified to homogeneity from conditioned medium by a two-column procedure which consisted of a Cu2+-chelating Sepharose column followed by TSK G3000SW gel filtration. The native molecular mass of rcFlc6S was estimated to be 102 kDa and the subunit size was 94 kDa. The kinetic properties of cGlc6S were similar to those of human Glc6S isolated from liver. rcGlc6S was endocytosed by fibroblasts from patients with mucopolysaccharidosis type IIID via the mannose 6-phosphate receptor-mediated pathway resulting in correction of the storage phenotype of these cells.
Keywords: Liver
Cells, Cultured
CHO Cells
Mucopolysaccharidosis III
Disease Models, Animal
Enzyme Precursors
Recombinant Proteins
Gene Expression
Genetic Vectors
Molecular Weight
DOI: 10.1042/bj3270089
Appears in Collections:Aurora harvest 5
Paediatrics publications

Files in This Item:
There are no files associated with this item.

Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.