重组人CK2β亚基的原核表达、纯化与鉴定

Prokaryotic Expression, Purification and Characterization of Recombinant Human Protein Kinase CK2β Subunit

将构建成功的人蛋白激酶CK2 β亚基cDNA的重组质粒 ,转化大肠杆菌BL2 1(DE3) ,在IPTG诱导下表达 .表达蛋白大多数以不溶形式存在 .6L (约 10 2g)表达菌抽提得到约 2 0mg的可溶性表达产物 ,通过P11磷酸纤维素一步层析分离 ,得到 6 8mg纯化蛋白 .SDS 聚丙烯酰胺凝胶电泳结果显示纯化的蛋白为一分子质量2 6ku的单一蛋白带 .蛋白质印迹结果证明 :纯化的表达产物与抗人CK2 β抗体可发生特异性免疫反应 .CK2 β亚基对CK2α有激活作用 ,纯化的CK2α和β亚基在等摩尔混合时即可组成有最大生物活性的全酶 .实验结果有力地证明了克隆表达与纯化的重组蛋白是人蛋白激酶CK2 β亚基 .

Protein kinase CK2 is a heterotetramer ser/thr protein kinase composed by two catalytic subunits (α or α′) and two regulatory subunits (β). The recombinant plasmid containing human CK2β subunit cDNA was transformed into E.coli BL21(DE3) and expressed induced by IPTG. Most of the expressed CK2β proteins were insoluble. From 6 L(about 10 4 g) bacteria, 20 mg soluble protein was extracted from the insoluble pellet and purified by a single step P11 phosphocellulose chromatography, the yield of purified CK2β subunit was 6 8 mg. SDS PAGE analysis of the purified protein showed only one band with molecular mass of 26 ku. Western blot analysis confirmed that the expressed product was human CK2β subunit. Addtion of the CK2β subunit to CK2α subunit led to maximum stimulation at a 1∶1 molar ratio of both subunits. These results demonstrated strongly that the cloned, expressed and purified recombinant protein was human CK2β subunit. The large amount of purified recombinant CK2β protein lays solid basis for further study directly the characteristics of the enzyme and the relationship of structure and function between CK2β subunit and its interacted proteins.