[ Objective] This study aimed to construct the His-tagged prokaryotic expression vectors harboring zinc finger domain （ZF） and central domain （ acidic domain ＆ zinc finger domain, CT） of MDM2, respectively, and p...[ Objective] This study aimed to construct the His-tagged prokaryotic expression vectors harboring zinc finger domain （ZF） and central domain （ acidic domain ＆ zinc finger domain, CT） of MDM2, respectively, and preliminarily identified biologic activity of the purified fusion proteins. [ Method ] ZF and CT cod- ing regions were amplified from MDM2 cDNA library by PCR and separately inserted into prokaryotie expression vector pET28b to construct the recombinant plas- mids. After verification by enzyme digestion, the recombinant plasmids were separately transformed into E. coli DH5c~ competent cells. The expressed recombinant plasmids were purified using Ni-NTA magnetic beads and identified by SDS-PAGE and Western blotting. [ Result] The molecular weight of His-ZF and His-CT fu- sion proteins was 21 and 31 kD, respectively. E Conclusion] Recombinant fusion proteins containing ZF and CT of MDM2 were obtained successfully, which laid the foundation for subsequent protein crystallization and three-dimensional structure analysis.展开更多
基金Supported by Project of Donghua UniversitySpecial Fund of Basic Research and Operating Expenses for Central Universities and Colleges in China(14D110511)
文摘[ Objective] This study aimed to construct the His-tagged prokaryotic expression vectors harboring zinc finger domain （ZF） and central domain （ acidic domain ＆ zinc finger domain, CT） of MDM2, respectively, and preliminarily identified biologic activity of the purified fusion proteins. [ Method ] ZF and CT cod- ing regions were amplified from MDM2 cDNA library by PCR and separately inserted into prokaryotie expression vector pET28b to construct the recombinant plas- mids. After verification by enzyme digestion, the recombinant plasmids were separately transformed into E. coli DH5c~ competent cells. The expressed recombinant plasmids were purified using Ni-NTA magnetic beads and identified by SDS-PAGE and Western blotting. [ Result] The molecular weight of His-ZF and His-CT fu- sion proteins was 21 and 31 kD, respectively. E Conclusion] Recombinant fusion proteins containing ZF and CT of MDM2 were obtained successfully, which laid the foundation for subsequent protein crystallization and three-dimensional structure analysis.
