Cloning,Expression of Human Nucleolus dUTPase and the Determination of Its Enzymatic Activity
CHEN Qiao lin 2) , REN Hong wei 1) , KANG Qiao hua 1), WANG Zong yuan 2) , RU Bing gen 1)*
( 1) Department of Biochemistry and Molecular Biology, College of Life Sciences, Peking University, National Laboratory of Protein Engineering, Beijing 100871, China; 2) College of Animal Husbandry and Veterinary Medicine, Yangzhou University, Yangzhou 225009,China
Abstract：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.
陈巧林,任宏伟,康巧华,王宗元,茹炳根. 人细胞核dUTPase的克隆表达及其酶学活性[J]. 中国生物化学与分子生物学报, 2003, 19(05): 606-611.
CHEN Qiao lin 2) , REN Hong wei 1) , KANG Qiao hua 1), WANG Zong yuan 2) , RU Bing gen 1)*. Cloning,Expression of Human Nucleolus dUTPase and the Determination of Its Enzymatic Activity. Chinese Journal of Biochemistry and Molecular Biol, 2003, 19(05): 606-611.
