Cloning And Expression Of Superoxide Dismutase Gene From Deinococcus Radiodurans In E.coli

Objective:In this research, the gene of Mn-SOD of Deinococcus radiodurans was cloned into an expression plasmid and the recombinant SOD fusion protein was expressed in E.coli in high efficiency. Then the protein was purified by metal-chelating affinity chromatography and the purified SOD was studied. These results are to provide a foundation for further studies on the radioresistant mechanism of Deinococcus radiodurans and for further development and application of SOD.Methods:1. Cloning the gene of Mn-SOD of Deinococcus radiodurans and constructing the recombinant plasmid pET-SOD. SOD gene was amplified by PCR from genomic DNA of Deinococcus radiodurans and inserted into expression plasmid pET-30a(+) to construct pET-SOD. The recombinant plasmid was transformed into cloning host E.coli DH5α. The positive clones were selected by using Kanamycin resistance, and recombinant plasmid was identified by enzyme digestion and sequence analysis.2. Expression and purification of the recombinant SOD fusion protein. The recombinant plasmid pET-SOD was transformed into expression host E.coli BL21(DE3). The recombinant protein was expressed by the induction of isopropyl-β -D-thiogalactopyranoside (IPTG) and was analyzed by SDS-PAGE. Then the SOD fusion protein was purified by metal-chelating affinity chromatography and the purified product was assessed by SDS-PAGE.3. Study on the properties of the purified SOD fusion protein. The purified SOD was analyzed by determining total protein and enzyme activity, identifying its category, detecting its ultraviolet absorption spectrum, and testing activity stability under different conditions.Results:1. The recombinant expression plasmid pET-SOD was constructed. And after double enzyme digestion, the plasmid vector fragment (5.4Kb) and target gene fragment (0.6—0.7kb) were shown respectively. Sequence analysis indicated that the target SOD gene was 633bp and the coding sequence of SOD fusion protein is 780bp, coding 260 amino acid residues. There is a one-base difference between the obtained sequence and the previously published sequence in TIGR or GenBank. The homology was 99%.2. The expression protein of SOD was induced by IPTG at 37°C. The 29kD protein was found after SDS-PAGE, accordant with anticipation. After purified by affinity chromatography, the SOD fusion protein showed almost a single band after SDS-PAGE.3. The purified SOD was identified to be Mn-SOD. Its specific activity reaches 19344U/mg protein. In the ultraviolet absorption spectrum, it shows specific absorption at 280nm. It can maintain high activity in a wide pH range and it is also stable to high temperature.Conclusion:The recombinant expression plasmid pET-SOD was constructed successfully. The SOD fusion protein was expressed effectively and found by SDS-PAGE. By affinity chromatography, the purified product could be obtained conveniently and fast with a high purity, high enzyme activity, and good stability. These results will provide theoretical and experimental foundation for further studies on the radioresistant mechanism of Deinococcus radiodurans and on establishing a convenient and efficient way to product Mn-SOD. This will be helpful for the development and application of SOD.