人细胞核dUTPase的克隆表达及其酶学活性

以阿尔茨海默病 (Alzheimer’sdisease ,AD)患者脑cDNA文库质粒为模板 ,用PCR方法扩增得到人细胞核dUTP焦磷酸酶 (dUTPase)的cDNA ,将其克隆到谷胱甘肽 S 转移酶 (GST)融合表达载体pGEX 4T 1中 ,并在大肠杆菌BL2 1中获得高效表达 .表达的融合蛋白GST dUTPase经过谷胱甘肽 Sepharose 4B亲和层析 ,凝血酶酶切和SephacrylS 10 0纯化 ,得到高纯度dUTPase蛋白 .通过SDS PAGE ,氨基酸组成分析 ,N端氨基酸序列测定以及HPLC测Mr 结果与期望值一致 .通过检测该酶水解dUTP释放的焦磷酸 (PPi)来测定表达产物dUTPase蛋白及GST dUTPase融合蛋白的酶活性 ,发现两蛋白都具有正常的酶水解dUTP活性 ,但融合蛋白的活性比dUTPase蛋白低 7～ 8倍 .同时研究了Mg2 +和EDTA对酶活性的影响

Cloning,Expression of Human Nucleolus dUTPase and the Determination of Its Enzymatic Activity

The cDNA of dUTP pyrophosphatase (dUTPase) in human nucleus was amplified by PCR from the cDNA library of the AD patient's brain, then cloned into the fusion expression vector of GST——pGEX 4T 1, and highly expressed in E.coli BL21. The fusion prottein GST dUTPase was purified by affinity chromatography, thrombin digestion and gel filtration on Sephacryl S100, and dUTPase was finally obtained. By detecting the quantity of PP i released from the hydrolyzation of dUTP, the activity of the recombined protein dUTPase and the fusion protein GST dUTPase was quantified. Both of them were found to have the activity of hydrolyzing dUTP, just like the normal enzyme. However, the activity of the recombined protein dUTPase was about 7～8 times higher than that of the fusion protein. Meanwhile, the effect of Mg 2+ and EDTA on the activity of enzyme was measured.