论文部分内容阅读
为了开辟苯丙酮尿症治疗新途径 ,本实验室构建了插入水稻苯丙氨酸脱氨酶 (PAL) c DNA的重组表达质粒 ,并用它转化大肠杆菌 BL2 1 DE3获得工程菌 BL2 1 DE3p ET2 8C-r PAL -1-c DNA.经异丙基硫代 -β-D-半乳糖苷诱导后 ,通过 SDS- PAGE及微型蛋白双向电泳 ,证明表达的目的蛋白的分子量为 78.6ku,主要存在于破碎菌体的沉积物中 (以包涵体形式存在 ) .按 Zucker法测定 PAL酶活性 ,并进行酶动力学实验 .测得表达蛋白的酶比活性为 462 nmol·g-1·min-1,Km,vmax分别为 0 .50 mmol·L-1和3. 0 5nmol · min-1.结果证明所建立BL2 1 DE3p ET2 8C-r PAL -1-c DNA大肠杆菌菌株能高效表达有活性的 PAL .
In order to open up a new way for the treatment of phenylketonuria, we constructed a recombinant expression plasmid inserted into rice phenylalanine ammonia-lyase (PAL) c DNA and transformed it into BL21 DE3p ET2 8C -r PAL -1-c DNA.After induced by isopropylthio-β-D-galactoside, the molecular weight of the expressed protein was 78.6ku by SDS-PAGE and two-dimensional electrophoresis of miniature proteins, The enzyme activity of PAL was determined by the Zucker method and the enzymatic activity was measured.The enzyme activity of the expressed protein was 462 nmol · g-1 · min-1, Km and vmax were 0.50 mmol·L-1 and 3.05 nmol · min-1, respectively. The results demonstrated that the constructed BL21 DE3p ET2 8C-r PAL-1-c DNA E. coli strain can express active PAL .