幽门螺杆菌PPK的纯化与功能分析

The Purification and Function Analysis of Helicobacter Pylori Polyphosphate Kinase

目的:表达纯化幽门螺杆菌多聚磷酸激酶,并测定其功能。方法:将幽门螺杆菌多聚磷酸激酶基因克隆入原核表达载体PQE80L中,在大肠杆菌(E.coli)DH5-α中表达。用BD Talon resin纯化目的蛋白。并在体外测定其合成多聚磷酸盐及转化多聚磷酸盐至ATP的能力。结果:成功构建了原核表达载体,得到高表达量的融合蛋白。经BD Talon resin纯化获得较高纯度的His-多聚磷酸激酶N端融合蛋白。体外实验证实该酶可以有效合成不同链长的多聚磷酸盐,并且在适当条件下可将多聚磷酸盐转化为ATP。结论:利用原核表达载体可很好表达幽门螺杆菌多聚磷酸激酶,纯化后的蛋白具有良好生物活性,是一个具备合成多聚磷酸盐及转化其为ATP的双向功能的酶。

Objective： To express and purify the polyphosphate kinase of Helicobacter pylori and analyze its function in vitro.Methods： The polyphosphate kinase gene of Helicobacter pylori were inserted into prokaryotic expression vector PQE 80L.The expression of the gene was induced in E.coli DH5-α.Then the 6-His fused protein was purified by using BD Talon resin.The activity of synthesizing polyphosphate and transfering polyphosphate to ATP of PPK were detected.Results： The expression of PPK was constructed and His-fused PPK was induced.The N-His fused protein was purified by BD Talon resin.This protein can synthesize Polyphosphate of different length or transfer polyphosphate to ATP under certain condition.Conclusions： PPK had good expression in prokaryotic expression vector PQE 80L and the protein showed biological activity of synthesizing polyphosphate and transferring polyphosphate to ATP.