DEK蛋白C末端DNA结合域的原核表达、纯化及其与DNA的结合活性

Prokaryotic Expression,Purification and DNA Binding Activity of DEK Protein's Carboxy-terminal DNA-binding Region

DEK蛋白C末端DNA结合域(简称CDB)是近年新发现的一个DEK蛋白与DNA的结合域,其中含有多个磷酸化位点,与DEK蛋白的功能密切相关。利用原核表达系统表达DEK蛋白的CDB肽段并进行纯化,具体为以pET-30a(+)为载体质粒,E.coliBL21(DE3)为宿主细胞,构建重组基因工程菌,以IPTG诱导目的蛋白的表达,用Ni-NTA纯化的重组蛋白样品来进行SDSPAGE电泳分析,约在10.7kDa处出现明显的特征蛋白条带。凝胶迁移分析证实DEK蛋白C末端DNA结合域与DNA的结合倾向于与超螺旋型DNA相结合,同全长的DEK蛋白与DNA的结合具有类似的特点,表明DEK蛋白C末端DNA结合域在DEK蛋白与DNA的结合中可能具有一定的作用。

DEK protein＇ s carboxy-terminal DNA-binding region （CBD） is a newly found DNA-binding domain of DEK, which contains several phosphorylation sites and has a close correlation with DEK protein＇s function in vivo and in vitro. Using prokaryotic expression system, the peptide of DEK protein＇s carboxy-terminal DNA-binding region （CDB） was expressed and purified. In detail, the CDB DNA fragment was constructed into pET-30a （ ＋ ） vector,and E. coli BI21 （DE3） competent cells were used as host cells. The fusion protein His- CBD was expressed by induction of IPTG and purified by Ni-NTA agarose. The result of SDS-PAGE showed that the molecular weight of the purified protein was about 10.7kDa. Electrophoretic mobility shift assay （EMSA） indicated that DEK-CDB prefered to bind to supercoiled form of DNA in vitro, it had similar character to the binding of whole length DEK protein with DNA. This suggested that the carboxy-terminal DNA-binding region of DEK protein might function on the binding of DEK protein to DNA partly.