Study On The Expression,Purification And Structure Of A Thermostable Iron Superoxide Dismutase

The thermostable FeSOD gene was separated from a new strain of Geobacillus. The lengthof this gene was1236bp, coding411amino acid residues. By bioinformatics analysis knew thatthe molecular weight of FeSOD was47.2kDa, and the isoeletric point was4.74.In order to explore the enzymology and structural properties of FeSOD, the FeSOD genewas cloned into fusion expression plasmid pET-28a, then transformed this recombinant plasmidto E.coli BL21(DE3) for expression under induction of IPTG. The identification results ofSDS-PAGE and Western blotting show that the target protein was successfully expressed. Theexpressed protein was purified by the process of His-tag affinity chromatography andanion-exchange chromatography.Through this two-step purification obtained the purity above95%of target protein. The purified protein was subjected to ultrafiltration and grop the bestconcentration to crystal. With a series of exploratory experiment, found the best crystal growthcondition, and obtained the FeSOD crystal with X-ray diffraction resolution of2.6. Byanalyzing the FeSOD crystal data, FeSOD structure was to be resolved. The results showed thatFeSOD is a tetramer, and each monomer contain8α-helix and3β-sheet, the active site of eachmonomer contain an active channel that formed by the β-sheet,an iron ions and three histidineresidues and an aspartic residues which are conncet to iron ions.In order to explore the heat-resistant mechanism of thermostable FeSOD. Compared thestructure of thermostable FeSOD, thermostable MnSOD and ordinary FeSOD together. Theresults showed that the structure difference between thermostable FeSOD and thermostableMnSOD were relative bigger, and there was a small different area between thermostable FeSODand ordinary FeSOD. For ordinary FeSOD the different area had a loop that separate the α-helix,but for thermostable FeSOD there was no loop, it，s completely composed by α-helix. Theα-helix is the most stable structure of the tertiary structure, while the loop is very unstablestructure. This may make the structure of ordinary FeSOD can be changed easily. So this regionmay determine the heat-resistant mechanism of thermostable FeSOD. Next we compared theamino acid sequences of ordinary FeSOD, thermostable FeSOD and thermostable MnSODtogether. We found that region two thermostable SOD commonly contain Y (tyrosine residues),but ordinary FeSOD corresponding contain F (phenylalanine residues). Therefore, we concludedthat the372site amino acids residues of thermostable FeSOD was key amino acids residues thatmay determine the heat-resistant mechanism of thermostable FeSOD. The next experiments, wemade thermostable FeSOD372site Y (tyrosine residues) mutated to F (phenylalanine residues),and found what would be change of mutant protein. This experiments completed the crystals of this enzyme, which will built a foundation forthe follow study on SOD strucyural biology mechanism and thermostable mechchanism.