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

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℃.