CRYSTALLIZATION OF RECOMBINANT SUPEROXIDE DISMUTASE ENZYME FROM Staphylococcus equorum

• Main Author: Puji Rahayu, Anis
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/45795
• Institution: Institut Teknologi Bandung

Superoxide dismutase (SOD) is an antioxidant enzyme that catalyzes the reduction of superoxide anion radicals into hydrogen peroxide (H2O2) and oxygen (O2). Recombinant SOD (rSOD) from Staphylococcus equorum has been characterized in Biotechnology Laboratory School of Pharmacy ITB, but it’s structure has not been determined. Crystallographic structure determination of a protein by X-ray diffraction needs 3D single crystal. Crystallization is the critical point for structural determination by X-ray crystallography. Quality of protein material and crystallization strategy is crucial for the success in obtaining the crystal. The quality of materials rSOD S. equorum was achieved upon stepwise purification using affinity chromatography on a nickel column and anion exchange chromatography. Copurified proteins were successfully separated in the anion-exchanger column at a very small conductivity difference, as near as 3 mS/cm. The rSOD suffered from physical instability such as aggregation and degradation upon storage at -20 °C. Screening for crystallization conditions was initiated by increasing the rigidity of protein molecule and precipitation test. rSOD structural rigidity was improved in the presence of MgCl2, CaCl2, ZnSO4, and Na2EDTA at a concentration range of 50 to 200 mM as suggested from the increase in its melting point observed using Thermofluor experiment. Determination of concentration and type of precipitant as well as protein concentration suitable for initial screening established the protein saturation and solubility profile. This directed approach resulted in suspected rSOD S. equorum crystals.