Mammalian Expression and Mass Spectrometric Characterization of Highly Active Human Arylsulfatase A.

Authors

T Pal, M Dabarera, A Dutta, G Smolenski, B D Dickson, A Williamson, W Kelton

Published in

Biotechnology journal. Volume 21. Issue 9. Pages e70312.

Abstract

The lysosomal enzyme Arylsulfatase A (ARSA) plays a critical role in hydrolyzing sulfatides from within cells. While gene therapies for ARSA replacement have recently been approved, challenges remain in recombinantly producing large amounts of active ARSA, especially for research purposes where generating stable cell lines is cumbersome. A major bottleneck is the need to convert the active-site residue Cysteine at position 69 to formylglycine by a formylglycine-generating enzyme (FGE). Further, transporter mannose-6-phosphate receptors (M6PR) are required to mediate active transport of ARSA out of the cell. To address these issues, we have optimized a three-plasmid transient transfection approach that produces ARSA with active site conversion of over 70% as measured by mass spectrometry and is highly active in assays with a model substrate. We find the optimal ARSA:M6PR:FGE plasmid ratio to be 8:1:1, which balances active site conversion with total protein yield. This optimized protocol will assist in producing highly active ARSA for biochemical studies in research labs with access to standard cell culture facilities.

PMID:
42788422
Bibliographic data and abstract were imported from PubMed on 25 Sep 2026.

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