丙酮酸磷酸双激酶基因克隆、表达及应用
Gene cloning，expression of pyruvate orthophosphate dikinase,purification and its appliacation
丙酮酸磷酸双激酶（PPDK）可催化ATP-荧光素-荧光素酶生物发光体系的产物之一AMP转化为ATP，在生物发光法检测细菌活细胞的实际应用中具有重要意义。本研究克隆到来源于天蓝色链霉菌（Streptomyces coelicolor）A3(2)的丙酮酸磷酸双激酶基因，并以pET28a(+)为载体质粒，E.coli BL21(DE3)为宿主细胞，构建基因重组菌E.coli BL21(DE3)（pET28a-ppdk）。经异丙基-β-D-硫代吡喃半乳糖苷诱导重组菌株过量表达，目的蛋白质经过亲和层析纯化后，将重组蛋白质样品经聚丙烯酰胺凝胶电泳分析，在97,000Da处出现显著的单一蛋白质条带。活性检测结果表明，该重组酶对于生成ATP的化学反应具有良好的催化活性。
he pyruvate phosphate dikinase catalyzes the deamination of AMP to ATP.The AMP was the production of the bioluminescence reaction system( ATP-luciferin-Luciferase ).This fluorescent method plays an important role in the application of bioluminescent living cells detection. We have cloned ppdk gene from Streptomyces coelicolor .The pET28a vector was for the plasmid，E.coliBL21(DE3)acted as the host cells,then the recombinant strains were constructed. The target protein was over expressed by IPTG induction.The molecular mass of ppdk enzyme was estimated to be 97,000 Da by gel filtration chromatography by SDS-PAGE. The result of activity detection reveals that the recombined enzyme has an excellent catalytic activity.
夏雨、弓紫丰、王周平
生物化学分子生物学生物工程学
食品检验丙酮酸磷酸双激酶克隆表达P生物发光
Food analysisppdkcloneexpressionATP-Bioluminescence
夏雨,弓紫丰,王周平.丙酮酸磷酸双激酶基因克隆、表达及应用[EB/OL].(2012-04-13)[2025-08-11].http://www.paper.edu.cn/releasepaper/content/201204-205.点此复制
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