牙龈卟啉单胞菌蛋白酶Ltp1的表达、纯化及酶学性质研究  

Expression,purification and enzymatic properties of protease Ltp1 from porphyromonas gingivalis

作　　者：顾前程 夏荣[1] 孔文文 Gu Qiancheng;Xia Rong;Kong Wenwen(Dept of Stomatology,The Second Affiliated Hospital of Anhui Medical University,Hefei 230601;Laboratory of Structure and Biochemistry,University of Science and Technology of China,Hefei 230601)

机构地区：[1]安徽医科大学第二附属医院口腔科,合肥230601 [2]中国科学技术大学结构与生物化学实验室,合肥230601

出　　处：《安徽医科大学学报》2020年第6期974-978,共5页Acta Universitatis Medicinalis Anhui

基　　金：安徽省自然科学基金(编号:1508085MH156)。

摘　　要：采用重组法构建Ltp1的原核表达载体,并将其转化至E.coli BL21(DE3)中进行异丙基-β-D-硫代半乳糖苷(IPTG)诱导过表达,然后用镍柱层析法粗纯化,分子筛细纯化得到目的蛋白,以SDS-PAGE电泳和Western blot分析表达产物;将目的蛋白和底物对硝基苯磷酸二钠显色反应分析其活性。SDS-PAGE结果显示有与目的蛋白理论相对分子质量一致的表达条带;Western blot分析证实该蛋白带有6个组氨酸(His)标签的目的蛋白,能够催化磷酸单酯的活性;计算出米氏常数(Km)和最大反应速率(Vmax)分别为70.4μmol、51.1μmol/(L·min),pH 5.0~6.0催化活性最高,在37~50℃具有较高活性。The prokaryotic expression vector of Ltp1 was constructed by recombinant method,and transformed into E.coli BL21(DE3)for over-expression induced by isopropyl-β-D-galactoside(IPTG),then the target protein was coarsely purified by nickel column chromatography and fine purified by molecular sieve,and the product was expressed by SDS-PAGE electrophoresis and Western blot.The activity of the target protein was analyzed by color reaction with disodium p-nitrophenyl phosphate.SDS-PAGE results showed that there was an expression band consistent with its theoretical relative molecular weight.Western blot analysis confirmed that the protein was indeed a target protein with six histidine(His)tagswhich could catalyze the activity of phosphate monoester.The Michaelis constant(Km)and the maximum reaction rate(Vmax)were calculated to be 70.4μmol and 51.1μmol/(L·min)respectively,which had the highest catalytic activity at pH 5.0~6.0 and 37~50℃.

关 键 词：牙龈卟啉单胞菌 Ltp1(PG1641) 蛋白表达纯化 牙周病 酶学研究 

分 类 号：R781[医药卫生—口腔医学]