活性SET7/9融合蛋白的表达与纯化

目的:表达与纯化活性GST-SET7/9融合蛋白(SET7/9融合蛋白).方法:以含人SET7/9基因结构域全长cDNA的pCMV-SET7/9质粒为模板,聚合酶链反应(PCR)法扩增获得目的基因片段,将测序正确的目的基因片段经EcoR Ⅰ与Xho Ⅰ双酶切,插入载体pGEX-6P-3中,构得质粒经转染和异丙基β-D硫代半乳糖苷(IPTG)诱导表达融合蛋白,纯化后采用组蛋白甲基化转移酶(HMT)测定法鉴定蛋白活性.结果:构成pGEX-SET7/9重组质粒,表达分子质量约为77 kD的融合蛋白,纯化后测得CPM值明显高于对照组.结论:pGEX-SET7/9可表达高活性的SET7/9融合蛋白.

Expression and purification of a functional SET7/9 fusion protein

Objective: To express and purify a functional fusion protein GST-SET7/9.Methods: pCMV-SET7/9 plasmid containing full-length human SET7/9 cDNA was used as a template for polymerase chain reaction(PCR).For correct reading code frame,the sequence-checked fragment was cleaved with EcoR Ⅰ and Xho Ⅰ,melted and linked with pGEX-6P-3 expression vector.Positive clone was selected from transformed E.coli cells and IPTG-induced for the expression of the fusion protein.The activity of purified protein was measured using histone methyltransferase(HMT) assay.Results: Recombinant plasmid pGEX-SET7/9 was constructed,expressing a fusion protein with the molecular mass of about 77 KD.The CPM value of the protein was much higher than the control.Conclusion: pGEX-SET7/9 can express active SET7/9 fusion protein in prokaryotic cells.