PRODUCTION AND CHARACTERIZATION OF RECOMBINANT SPARC, A CYSTEIN-RICH MULTIDOMAIN PROTEIN

FEDERICO PRADA; LEONARDO ALONSO; EDGARDO SALVATIERRA; GONZALO DE PRAT GAY; OSVALDO PODHAJCER; ANDREA S. LLERA

Buenos Aires

Congreso; International Union of Pure and Applied Biophysics Congress; 2002

SPARC (Secreted Protein, Acidic and Rich in Cysteins) is a multidomain glycoprotein belonging to the “natively unfolded” family of proteins, that modulates interactions between cells and extracellular matrix. SPARC inhibits cell spreading, induces rounding of cultured endothelial cells and disassembles focal adhesions. In malignant tumors SPARC proved to play a crucial role in tumorigenicity. However, the molecular mechanisms that underlie these processes are not clear, and there is no evidence of the nature of putative receptor/s that may interact with SPARC.We have cloned and expressed human full-length SPARC for studying its biochemical characteristics, mapping some of its biological activities and searching for putative ligands. In order to validate this protein, we have also purified the native protein from conditioned media of A375N human melanoma cells. SPARC expressed as inclusion bodies in E.coli rendered a protein prone to agregation that did not show biological activity. We therefore expressed SPARC as a secreted protein, in suspension cultures of transfected Drosophila S2 cells. S2- and A375N- derived SPARC were purified by anionic exchange and gel filtration in similar conditions, and proved to have identical circular dichroism spectra between 190-260 nm. We are currently testing SPARC biochemical characteristics and biological activity in different cell lines.