【摘要】 目的:研究重组小鼠CK2α′亚基在大肠杆菌中的谷胱甘肽S转移酶(GST)融合表达和纯化后活性的测定.方法:将含有小鼠CK2α′cDNA的pGEXGST-MCK2α′重组质粒转化大肠杆菌BL21,异丙基硫代半乳糖苷(IPTG)诱导表达,采用谷胱甘肽-琼脂糖珠4B柱纯化,SDS-聚丙烯酰氨凝胶电泳银染鉴定.将纯化的CK2α′与CK2β以不同的摩尔比混合,找到构成有完全活性的CK2全酶的最佳摩尔比.用各种已知CK2底物鉴定重组小鼠CK2全酶的性质.对纯化的CK2α′和重组CK2全酶进行动力学分析.结果:重组质粒转化表达菌经诱导后出现Mr约为38ku过度表达蛋白,约占菌体总蛋白的31.1%.SDS-聚丙烯酰氨凝胶电泳银染显示纯化的蛋白为单一蛋白带.纯化的CK2α′和β亚基等摩尔比混合可组成有完全活性的全酶.重组CK2全酶的性质与天然CK2一致.重组CK2全酶对ATP或GTP的米氏常数(Km)值均小于单独CK2α′,而最大反应速度(Vmax)值均大于单独CK2α′.结论:重组蛋白是小鼠蛋白激酶CK2α′亚基.更多还原

【Abstract】 AIM:To study the glutathione S-transferase(GST)fusion expression,purification and activity assay of recombinant mouse protein kinase CK2α’ subunit in Escherichia coli.METHODS:The recombinant plasmid containing mouse CK2α’ subunit cDNA was transformed into Escherichia coli BL21 and specifically induced by IPTG,purified with glutathione Sepharose 4B affinity chromatography.The purified recombinant protein was analysed by silver-stained SDS-PAGE.The pure CK2α’ and β subunit proteins were mixed at different molar ratios to discover the optimum molar ratio so that recombinant holoenzyme CK2 could display full activity.Recombinant CK2 holoenzyme was characterized by the known substrates of CK2.The pure CK2α’ and recombinant holoenzyme CK2 were studied through kinetic analysis.RESULTS:One protein with molecular mass of 38 ku was overexpressed by ITPG induction.The fusion protein was composed of approximately 31.1% of the total bacterial proteins.Silver-stained SDS-PAGE analysis of the purified recombinant protein showed only one band.The pure mouse CK2α’ and β subunits mixed at the equal molar ratio displayed the full activity.Characteristics of recombinant CK2 holoenzyme was in agreement with native CK2.Recombinant CK2 holoenzyme showed a lower value of Km for ATP or GTP than CK2α’ alone,but a larger value of Vmax than CK2α’ alone.CONCLUSION:The recombinant protein is mouse protein kinase CK2α’ subunit.更多还原