Efficient one-step chromatographic purification and functional characterization of recombinant human Saposin C

Marialetizia Motta, Massimo Tatti, Simone Martinelli, Serena Camerini, Susanna Scarpa, Marco Crescenzi, Marco Tartaglia, Rosa Salvioli

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish
Pages (from-to)209-215
Number of pages7
JournalProtein Expression and Purification
Issue number2
Publication statusPublished - Aug 2011


  • Affinity chromatography
  • Endocytosis
  • Mass spectrometry
  • Overexpression
  • Protein-anionic phospholipids interactions
  • Sphingolipid activator protein

ASJC Scopus subject areas

  • Biotechnology


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