title: Efficient one-step chromatographic purification and functional characterization of recombinant human Saposin C.
abstract: Saposin (Sap) C is a small lysosomal disulfide bridge-containing glycoprotein required for glucosylceramide (GC) hydrolysis by glucosylceramidase (GCase). Sap C deficiency causes a variant form of Gaucher disease (GD), a rare genetic disorder characterized by GC accumulation in lysosomes of monocyte/macrophage lineage. Efforts to develop fast and efficient methodologies to express and purify Sap C have been made in the last years. Here, human Sap C was expressed in a bacterial strain that greatly enhances disulfide bond formation, and the recombinant protein was purified in a single chromatographic step using an affinity tag-based protein purification system. Mass spectrometry analysis demonstrated that disulfide bridges required for Sap C stability and functionality were retained. Consistently, the recombinant protein was shown to interact with anionic phospholipids-containing vesicles, and reconstitute GCase activity in vitro. Recombinant Sap C was efficiently endocytosed by Sap C-deficient fibroblasts, and targeted to lysosomes. These findings document that the bacterially purified Sap C exerts biological properties functionally equivalent to those observed for the native protein, indicating its potential use in the development of therapeutic intervention.
authors.affiliation: Department of Haematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.
authors.forename: Marialetizia
authors.initials: M
authors.lastname: Motta
authors.forename: Massimo
authors.initials: M
authors.lastname: Tatti
authors.forename: Simone
authors.initials: S
authors.lastname: Martinelli
authors.forename: Serena
authors.initials: S
authors.lastname: Camerini
authors.forename: Susanna
authors.initials: S
authors.lastname: Scarpa
authors.forename: Marco
authors.initials: M
authors.lastname: Crescenzi
authors.forename: Marco
authors.initials: M
authors.lastname: Tartaglia
authors.forename: Rosa
authors.initials: R
authors.lastname: Salvioli
mesh_terms: D015153:Blotting, Western; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D005347:Fibroblasts; D006801:Humans; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D049231:Saposins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D049231:Saposins