Expression And Trans-membrane Activity Of Human Cu, Zn-SOD-TAT PTD

Objective To construct the recombinant vector which can express human Cu,Zn-SOD-TAT PTD in E.coli, express and purify the target protein in vitro and study the trans-membrane activity of human Cu,Zn-SOD-TAT PTD. Methods Human Cu,Zn-SOD gene was amplified by PCR from human lung cDNA library and inserted into pUC19 and identified by sequencing. The sequenced plasmid was used as the template for PCR amplification of Cu,Zn-SOD-TAT PTD fusion gene with a 3'-primer containing TAT PTD coding frame,then the Cu,Zn-SOD-TAT PTD fusion gene was inserted into pUC19.The positive clones were proved by restrictive endonuclease mapping analysis and DNA sequencing. The Cu,Zn-SOD gene and Cu,Zn-SOD-TAT PTD fusion gene were sub-cloned into temperature-inducing expression vector pJW2 and the recombinant plasmids were transformed into E. coli DH5 a , respectively. The SOD and SOD-TAT PTD proteins were chiefly expressed as inclusion bodies.After inclusion bodies extraction,protein renaturing and icon-exchange chromatography,the biological activities of the recombinant proteins were tested.Treated with the purified SOD and SOD-TAT PTD proteins for 1.5h,the WISH cells were harvested and extracted to perform SOD enzyme assays. Results By restrictive endonuclease mapping analysis and DNA sequencing,we demonstrate that the constructed plasmids were the recombinant ones.The results of SDS-PAGE and western blot showed the nascent band of recombinant protein was obviously appeared with a molecular weight of 19KD and 22KD,respectively, and recombinant proteins could be specifically recognized by monoclonal antibody against human Cu,Zn-SOD. Biossay in vitro showed that specific activity of purified rhCu,Zn-SOD and rhCu,Zn-SOD-TAT PTD were 12 X 103NU/mg and 9.9 X 103NU/mg respectively. The biological activity of the WISH cells treated with SOD-TAT PTD protein washigh than those treated with SOD protein. Conclusion The expressed plasmidpJW2-SOD-TAT PTD was constructed. rhCu,Zn-SOD-TAT PTD was expressed inE.coli and pufified well. Comparied with rhCu,Zn-SOD,the fusion proteinrhCu,Zn-SOD-TAT PTD can translocate through cell membrane and enter culturedWISH cells.