title: Expression and purification of a human, soluble Arylsulfatase A for Metachromatic Leukodystrophy enzyme replacement therapy.
abstract: The production of active Arylsulfatase A is a key step in the development of enzyme replacement therapy for Metachromatic Leukodystrophy. To obtain large amounts of purified Arylsulfatase A for therapeutic use, we combined a retroviral expression system with a versatile and rapid purification protocol that can easily and reliably be adapted to high-throughput applications. The purification method consists of an initial ion-exchange DEAE-cellulose chromatography step followed by immuno-affinity purification using a polyclonal antibody against a 29-mer peptide of the Arylsulfatase A sequence. Immuno-adsorbed protein was eluted with a combination of acidic pH and an optimal concentration of the 29-mer peptide. This protocol reproducibly yielded approximately 100 microg of >99% pure human Arylsulfatase A, corresponding to 152 mU of enzyme activity, per liter of culture medium with properties similar to those of human non-recombinant protein.
authors.affiliation: Dipartimento di Scienze Biochimiche e Biotecnologie Molecolari, University of Perugia, Via del Giochetto, Italy.
authors.forename: Sabata
authors.initials: S
authors.lastname: Martino
authors.forename: Antonella
authors.initials: A
authors.lastname: Consiglio
authors.forename: Cristina
authors.initials: C
authors.lastname: Cavalieri
authors.forename: Roberto
authors.initials: R
authors.lastname: Tiribuzi
authors.forename: Egidia
authors.initials: E
authors.lastname: Costanzi
authors.forename: Giovanni Maria
authors.initials: GM
authors.lastname: Severini
authors.forename: Carla
authors.initials: C
authors.lastname: Emiliani
authors.forename: Claudio
authors.initials: C
authors.lastname: Bordignon
authors.forename: Aldo
authors.initials: A
authors.lastname: Orlacchio
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002457:Cell Extracts; D002460:Cell Line; D002478:Cells, Cultured; D002553:Cerebroside-Sulfatase / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism* / Q000627:therapeutic use*; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006801:Humans; D007525:Isoelectric Focusing; D007966:Leukodystrophy, Metachromatic / Q000201:enzymology* / Q000628:therapy*; D051379:Mice; D018345:Mice, Knockout; D009836:Oligodendroglia / Q000166:cytology; D011994:Recombinant Proteins / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D012995:Solubility; D013379:Substrate Specificity; D014161:Transduction, Genetic
chemical_list: D002457:Cell Extracts; D011994:Recombinant Proteins; D002553:Cerebroside-Sulfatase