蛋白酪氨酸磷酸酶SHP-1/SHP-2催化活性域的表达和动力学分析  

Expression and kinetic analysis of catalytic domain of protein tyrosine phosphatases SHP-1/SHP-2

作　　者：莫毅[1,4] 王伟[2] 梁方方 傅冠元[2] 蒋和生[1] Wayne Zhou 

机构地区：[1]广西大学动物科学技术学院,南宁530004 [2]美国路易斯安那州立大学生命科学院,巴吞鲁日LA70803 [3]广西畜牧研究所,南宁530001 [4]广西人口和计划生育研究中心,南宁530021

出　　处：《生物工程学报》2009年第2期274-278,共5页Chinese Journal of Biotechnology

摘　　要：为了分离纯化SHP-1/SHP-2催化活性域蛋白(分别命名为D1C/D2C),并估测其动力学常数,将已经构建好的D1C/D2C重组质粒转化Escherichia coli BL21菌株,经IPTG诱导表达、菌体裂解缓冲液悬浮和超声波破碎后,通过HPLC分离纯化D1C/D2C蛋白,所得产物进行SDS-PAGE电泳检测。然后,以pY作为去磷酸化反应的底物,利用孔雀绿显色法,通过双倒数作图法对纯化的D1C/D2C蛋白进行动力学分析。结果表明,本试验已成功地表达了D1C和D2C蛋白,主要以可溶性蛋白的形式表达;利用HPLC技术可有效地对D1C/D2C蛋白进行分离纯化;D1C的相对分子质量为34.6 kD,米氏常数Km=2.04 mmol/L,催化常数Kcat=44.98 s-1,特异性常数Kcat/Km=22.05 L/(mmol.s);D2C的相对分子质量为35.3 kD,米氏常数Km=2.47 mmol/L,催化常数Kcat=27.45 s-1,特异性常数Kcat/Km=11.11 L/(mmol.s);D1C的磷酸酶活性较强于D2C。In order to express and purify the catalytic domain of SHP-1/SHP-2 （named as D1C and D2C respectively） and determine their kinetics, the constructed D1C and D2C plasmids were transformed into Escherichia coli BL21 and the expression was induced with IPTG. The harvested cells were suspended in extraction buffer. After sonication, the solution was applied to HPLC and the results were confirmed by SDS-PAGE. The purified peptides were further subjected to kinetic specificity study using synthetic phosphotyrosine （pY） as substrate by malachite green method and analyzed by Lineweaver-Burk plot calculation. From this study, we found D1C and D2C were expressed successfully in soluble state in Escherichia coli BL21 and purified efficiently with HPLC system. The molecular weight of D1C was 34.6 kD, and its Michaelis constant （Km） was 2.04 mmol catalytic constant （Kcat） was 44.98 s-i, specific constant （Kcm/Km） was 22.05 L/（inmol · s）; the molecular weigh of D2C was 35.3 kD, and its Michaelis constant （Kin） was 2.47 mmol, catalytic constant （gcat） was 27.45 s^-1, specific constant （Kcm/Km） was L/（mmol ·s）. The enzyme activity of D1C is stronger than that of D2C.

关 键 词：SHP-1 SHP-2 蛋白表达 蛋白纯化 动力学分析 

分 类 号：Q55[生物学—生物化学]