蛋白酪氨酸磷酸酶shp-1催化活性域的纯化和动力学分析

Purification and kinetic analysis of catalytic domain of protein tyrosine phosphatases shp-1

为了获得分离纯化shp—1催化活性域蛋白（D1C）并估测其动力学常数，试验选择D1C的转化菌株经IVFG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蛋白进行动力学分析。结果表明：试验成功地表达了D1C蛋白，其分子质量约为34．6ku，主要以可溶性蛋白的形式表达；通过HPLC技术可有效地对D1C蛋白进行分离纯化；D1C蛋白的米氏常数Km=2．04mmol／L，催化常数（kcat）=44．98s^-1，特异性常数（kcat／Km）=22．05L／（mmol·s）。

To express and purify the catalytic domain of shp - 1 （ name as D1C ） and calculate its kinetic constants. We first expressed DI C from transformed E. coli BL21 bacteria under IPTG induction and purified D1C peptides through HPLC then confirmed by SDS -PAGE. The purified peptides were further subjected to kinetic specificity study using synthetic pY as substratc by malachite green method and analyzed by Lineweaver- Burk plot calculation. From this study, we found D1C peptides were induced to express with soluble state in E coli. BL 21 successfully and purified with HPLC system efficiently. The molecular weigh of D1C was 34.6 ku, and its michaelis constant （ Km ） was 2.04 mmol/L, catalytic constant （kcat） was 44.98 s ^- 1, specific constant （kcat/Km） was 22.05 L/（ mmol · s）.