{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T15:03:08Z","timestamp":1790348588082,"version":"4.1.0"},"reference-count":27,"publisher":"Wiley","issue":"9","license":[{"start":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T00:00:00Z","timestamp":1790294400000},"content-version":"vor","delay-in-days":24,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T00:00:00Z","timestamp":1788220800000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biotechnology Journal"],"published-print":{"date-parts":[[2026,9]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>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\u2010site residue Cysteine at position 69 to formylglycine by a formylglycine\u2010generating enzyme (FGE). Further, transporter mannose\u20106\u2010phosphate receptors (M6PR) are required to mediate active transport of ARSA out of the cell. To address these issues, we have optimized a three\u2010plasmid 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.<\/jats:p>","DOI":"10.1002\/biot.70312","type":"journal-article","created":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T10:48:15Z","timestamp":1790333295000},"source":"Crossref","is-referenced-by-count":0,"title":["Mammalian Expression and Mass Spectrometric Characterization of Highly Active Human Arylsulfatase A"],"prefix":"10.1002","volume":"21","author":[{"ORCID":"https:\/\/orcid.org\/0009-0001-9311-217X","authenticated-orcid":false,"given":"T.","family":"Pal","sequence":"first","affiliation":[{"name":"Te Aka M\u0101tuatua School of Science University of Waikato  Hamilton New Zealand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"M.","family":"Dabarera","sequence":"additional","affiliation":[{"name":"Te Aka M\u0101tuatua School of Science University of Waikato  Hamilton New Zealand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"A.","family":"Dutta","sequence":"additional","affiliation":[{"name":"Te Aka M\u0101tuatua School of Science University of Waikato  Hamilton New Zealand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0544-2687","authenticated-orcid":false,"given":"G.","family":"Smolenski","sequence":"additional","affiliation":[{"name":"MS3 Solutions, Ruakura Research Centre  Hamilton New Zealand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6860-903X","authenticated-orcid":false,"given":"B. 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