Construction,expression and purification of a thermostable,high yield recombined hCu,Zn-SOD mutant

Aim To obtain the rhCu,Zn-SOD mutant with high thermostability and yield,rhCu,Zn-SOD cDNA was modified by overlap PCR;and to establish the purification method of the rhCu,Zn-SOD mutant protein.Methods The Cys6 and Cys111 of hCu,Zn-SOD gene were mutated into Ala6 and Ser111 by overlap PCR.Then,the gene was cloned into vector pET22b(+),and transfered to BL21(DE3).After induced with IPTG,the expression of the mutant protein was detected by SDS-PAGE and western blot.The purification method of the mutant protein was established,and the purity of the mutant protein was detected by HPLC.Finally,the activity and thermostablity of the rhCu,Zn-SOD mutant was compared with the wide rhCu,Zn-SOD.Results The expression vector of the rhCu,Zn-SOD mutant was constructed sucessfully.The mutant protein was expressed in BL21(DE3) by IPTG induction,and the special expressed accounts for 64.6% of the total protein of the bacteria,and possesed antigen specificity with the mouse anti-human SOD polyclonal antibody.The rhCu,Zn-SOD mutant protein was purified,the purity is over 95%.The total amount active unit of mutant protein from 1g wet Ecoli.cells is about 2 times that of wild hCu,Zn-SOD.Meanwhile,the thermostability of the mutant is improved significantly.Conclusion The rmhCu,Zn-SOD6Ala,111Ser mutant with better thermostability and high yield was constructed;a relatively simple protein purification process of wide application value was worked out.