Expression and Purification of Zinc Finger Domain and Central Domain of MDMD2
Expression and Purification of Zinc Finger Domain and Central Domain of MDMD2作者机构:Donghua University Shanghai 201600 China
出 版 物:《Agricultural Biotechnology》 (农业生物技术(英文版))
年 卷 期:2016年第5卷第2期
页 面:37-39页
学科分类:0710[理学-生物学] 07[理学] 08[工学] 09[农学] 071007[理学-遗传学] 0901[农学-作物学] 0836[工学-生物工程] 090102[农学-作物遗传育种]
基 金:Supported by Project of Donghua University Special Fund of Basic Research and Operating Expenses for Central Universities and Colleges in China(14D110511)
主 题:Zincfinger domain Central domain MDM2 Prokaryotic expression Protein purification
摘 要:[ 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.