变形杆菌属脂肪酶LipK107的分离纯化、结晶及初步晶体学分析

Purification,crystallization and preliminary crystallographic analysis of a lipase from Proteus sp.K107

经过异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导,重组大肠杆菌高效表达出带有组氨酸标签的可溶性脂肪酶LipK107。通过亲和层析和凝胶层析两步法纯化,得到电泳纯的脂肪酶样品。在获得的纯酶基础上,进行了圆二色谱实验,以分析脂肪酶LipK107的二级结构组成。接着,使用悬滴气相扩散法进行了纯酶的结晶实验,经过晶体生长条件的初步筛选及优化,在0.1mol/L pH6.2柠檬酸钠,10%v/v异丙醇,15%w/v聚乙二醇4000条件下得到高质量的脂肪酶晶体,使用同步辐射光源收集了一套分辨率为2.0的X-射线衍射数据。

After induced by IPTG,the recombinant E.coli expressed Lipase LipK107 as HisX6 fusion protein.High purity enzyme was obtained by affinity chromatography and gel filtration chromatography.Purified lipase was analyzed by circular dichrosim（CD）in order to reveal the secondary structure composition of lipase LipK107.Then,purified lipase was crystallized by the hanging-drop vapour-diffusion method.High quality crystals were obtained using 0.1mol/L sodium citrate tribasic dihydrate pH6.2,10% v/v 2-propanol,15% w/v polyethylene glycol 4000 as precipitant.X-ray diffraction data was collected to 2.0 using synchrotron radiation.