Improvements in the purification and crystallization of Yarrowia lipolytica Sterol Carrier Protein-2.

PEREZ DE BERTI, FEDERICO J.; BURGARDT, NOELIA I.; FERREYRA, RAUL G.; ERMÁCORA, MARIO R.

Salta

Congreso; XXXIX Reunión Anual de la Sociedad Argentina de Biofísica. 3era Reunión de la Sociedad Latinoamericana de Proteínas.; 2010

Sociedad Argentina de Biofísica. Sociedad Latinoamericana de Proteínas.

The sterol carrier protein-2 (SCP-2) is found in the three super kingdoms of life, and it has been implicated in the transport and metabolism of lipids. SCP-2 is able to bind a vast number of ligands, and on the other side, it is known that it interacts with membranes through its N-terminal alpha helix. This differential interaction of SCP-2 with different ligands or membranes confirms the great structural plasticity of this family of proteins. Despite all the above information is not yet clear the general biological function of SCP-2. We previously have shown that Y. lipolytica sterol carrier protein-2 (YLSCP-2) is a 128 amino acid protein inducible by fatty acids and binds a variety of lipids1 (pI=10.2, PM=13903)(1). The aim of this work was to further explore the biochemical properties and structure of YLSCP-2. Recombinant YLSCP-2 was expressed in E. coli as inclusion bodies. After a single ionic-exchange chromatography of inclusion bodies dissolved by 8 M urea, YLSCP-2 was obtained highly pure, and its refolding was accomplished by dialysis. After dialysis a molecular exclusion chromatography was carried out in a Superdex G-75. The molecular exclusion chromatography separates a SCP-2 monomer from a SCP-2 dimer (as revealed by light scattering). Near- and far-UV CD and thermal unfolding experiments have shown that the SCP-2 monomer is partially unfolded. In order to avoid monomer formation the ionic-exchange chromatography was performed at pH 5.5. We also have observed that the dimer is more stable if stored at pH 5.5. Crystallization trials with the recombinant protein were performed, both, with and without ligands (palmitate), using the hanging-drop method. A variety of crystals were obtained, which constitutes a big improvement over our previous reports. These results should allow undertaking the structural characterization of the protein and their complexes with atomic resolution by X diffraction. (1)Ferreyra et. al. Arch. Biochem. Biophys. 453(2): 197-206 (2006).