SKRINING KONDISI KRISTALISASI ENZIM SUPEROKSIDA DISMUTASE REKOMBINAN DARI STAPHYLOCOCCUS EQUORURN
Superoxide dismutase is an enzyme that catalyzes the reduction of superoxide anion radicals into hydrogen peroxide (H202) and oxygen (02). SOD was found in most aerobic organisms, ranging from bacteria to mammals as to protect against reactive oxygen species. A synthetic gene encoding SOD of Staphyl...
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| Format: | Final Project |
| Language: | Indonesia |
| Online Access: | https://digilib.itb.ac.id/gdl/view/64301 |
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| Institution: | Institut Teknologi Bandung |
| Language: | Indonesia |
| Summary: | Superoxide dismutase is an enzyme that catalyzes the reduction of superoxide anion radicals into hydrogen peroxide (H202) and oxygen (02). SOD was found in most aerobic organisms, ranging from bacteria to mammals as to protect against reactive oxygen species. A synthetic gene encoding SOD of Staphylococcus equorum (sod4) has been designed and expressed in the Laboratory of Pharmaceutical Biotechnology, School of Pharmacy ITB. Overexpression of SOD gene in Escherichia coli BL21(DE3) produced recombinant superoxide dismutase (rSOD) of 23,47 kDa in size. Determination of three- dimensional structure of SOD S. equorum has not been previously done. This study was aimed to determine the optimal condition for the formation of protein crystals of rSOD to meet high resolution X-ray diffraction analysis requirement in order to determine the structure. The rSOD was purified using nickel-NTA affinity chromatography column with gradual elution using LEW buffer pH 8 containing increasing concentrations of imidazole. Removal of imidazole in the purified fractions was done using a dialysis membrane with a cutoff of 2 kDa and then the activity of rSOD was determined. The rSOD was concentrated using Macrosep and Nanosep, both with 10 kDa cutoff. The screening of rSOD S. equorum crystallization conditions was done using sitting-drop vapor diffusion method using Hampton Crystal Screen kit NatrixTM. Crystal formation was observed by light and polarization microscopes. Protein crystals were obtained in five NatrixTM reagent composition. Suitable condition for crystal formation was obtained in reagents with compositions of 0.1 M potassium chloride, 0.025 M magnesium chloride hexahydrate; 0.05 M sodium cacodylate trihydrate pH 6.0, and 15% v / v 2-propanol. |
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