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.
Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
