小麦细胞周期蛋白基因CDC25的原核表达及蛋白纯化

Prokaryotic Expression and Protein Purification of the Wheat CDC25 Gene

细胞分裂周期蛋白CDC25是一类苏氨酸/酪氨酸双特异性的磷酸酶,在动物上通过激活CDK进而推动细胞周期进程,然而植物CDC25的这一功能可能已经不重要或已被替代,故有关植物CDC25的功能还有待阐明。本研究构建了小麦CDC25基因Ta CDC25.1-1AL的原核表达载体;导入大肠杆菌的Ta CDC25.1-1AL基因能够被IPTG诱导表达,表达重组蛋白的表观分子量与理论分子量基本一致;利用Ni柱亲和层析方法获得了纯度较高的重组蛋白,为今后通过体外酶活分析、抗体制备和Western杂交等方法深入研究该蛋白和基因的功能奠定了基础。

Cell division cycle 25（CDC25） is a dual-specificity threonine/tyrosine phosphatase, which, in animals, promotes cell cycle progression through activating cyclin-dependent kinases（CDKs）. However, the role of plant CDC25 in cell cycle regulation may be nonessential or has been replaced by other factors. Therefore, the function of plant CDC25 remains to be elucidated. In this study, a prokaryotic expression vector for the wheat CDC25 gene Ta CDC25.1-1 AL was constructed and transformed into E. coli. Results from SDS-PAGE showed that Ta CDC25.1-1 AL could express efficiently in E. coli through IPTG induction. The molecular weight of the recombinant protein is consistent with its predicted molecular weight. Recombinant proteins were further purified by the Ni-column affinity chromatography method. The obtaining of purified Ta CDC25.1-1 AL recombinant proteins laid a foundation for the future studies of in vitro enzyme activity, antibody preparation and western blot analysis on Ta CDC25.1-1 AL.