Fe-sod Gene Cloning And Expression

Superoxide dismutase (EC 1.15.1.1), one kind of metalloenzyme, which has been found ubiquitously among oxygen-metabolizing organisms. According to the requirement of the metal species at the active site of the enzymes, superoxide dismutase has been classified four type: Cu/Zn-SOD, Mn-SOD, Fe-SOD and Ni-SOD. It could catalyse the dismutation of superoxide anion radical to molecular oxygen and hydrogen peroxide. Furthermore, superoxide dismutase has relation to many human diseases , so it has great therapeutic potential. Considering this point it has fine foreground of development and application.For the moment mostly all the SOD are separated and purificated from organism in nature. Accouting to the fact, people have obtained artificial SOD by gene technology in Amarica , Japan, England and Germany et al. While many kinds of Cu/Zn-SOD and Mn-SOD has been cloned and expressed in China, the Fe-SOD is different.In this paper, it is studied in clone and expression of the Fe-SOD gene from Nostoc commune A. The Fe-SOD gene was obtained from the genome DNA of Nostoc commune through PCR techniques, and cloned into pUCm-T vector. Its sequence was determined by DNA sequencing system. The sequence obtained from Nostoc commune A is not the same to that from Nostoc commune DRH1. Perhaps it comes from a different Nostoc subspecies. The probable super configuration of Fe-SOD has two domains similarly to the Fe-SODA. The expression vector contains genes that ecoding a peIB leader , Fe-SOD and a 6 X His tag which can give a facility for purification by nickel affinity chromatography. The constructed expression vector was subsequently inserted into with the DNA fragment, which was generated by PCR reaction with the pUCm-S plasmid as template, into IPTG-inducible expression vector pET22b(+) plasmid. The sequence of Fe-SOD gene in the expression vector was verified by DNA sequencing anlysis. High-yield expression of Fe-SOD fused protein was achieved in E.coli transformed with expression vector, and most of the Fe-SODexisted in the inclusion body after IPTG induction. It has a perfect expression in 37癈 environment inducted by 1mM IPTG in five hours. 78 percent of protein in E.coli BL21(DE3) is the Fe-SOD fused protein. The washed inclusion body was solublized with 8 M urea and purified by nickel affinity chromatography. The purified Fe-SOD was refolded by dilutedness in refolding solution with L-arginine. The activity of refolded Fe-SOD was determined by method of pyrogallie aid .The results showed that protein has superoxide dismutase active.