[Sited-directed mutagenesis of hCu, Zn-SOD gene and its expression in Synechococcus sp. PCC7942].
The Cys111 genetic code of human copper/zinc superoxide dismutase (hCu, Zn-SOD) gene in the pESOD plamid was mutated into the Ala111 code with site-directed mutagenesis, and then the plamid pESODT111 which contained groESL promoter, mutated hCu, Zn-SOD gene, rbcS-polyA terminator and reporter gene (Kanr) was constructed and transduced into Synechococcus sp. PCC7942 with homologous recombination platform. The results of PCR and DNA sequence analysis showed that the target nucleotide had been genetically integrated into genome DNA of the host cell. SDS-PAGE, Western blot and Pyrogallol autoxidation assay confirmed that the transformant strains expressed the mutated hCu, Zn-SOD protein. And the level of the mutated hCu, Zn-SOD protein reached a value of 3.61% of the total soluble protein. Furthermore, the transformants still retained 95% activities of SOD after 30 minutes at 80 degrees C environment, it indicated that the mutated hCu, Zn-SOD protein could endure higher temperature than the natural one.
Zhou
Zan-hu
ZH
School of Life Science, Xiamen University, Xiamen 361005, China. zzh_523@163.com
Zhang
Hai-yan
HY
Liu
Ren-hai
RH
Zhang
Jun
J
Zhou
Ke-fu
KF
D015153:Blotting, Western; D003300:Copper; D015870:Gene Expression; D006801:Humans; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D013482:Superoxide Dismutase / Q000235:genetics*; D046940:Synechococcus / Q000378:metabolism*; D015032:Zinc
D003300:Copper; D013482:Superoxide Dismutase; D015032:Zinc