由BL21DE3~(pET28C-rPAL-1-cDNA)高效表达具有酶活性的苯丙氨酸脱氨酶

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 .

High-level expression of phenylalanine ammonia lyase with enzyme activity in E.coli by BL21DE3~( pET28C-rPAL-1-cDNA)

In order to create an alternative treatment of phenylketonuria, recombinant expression plasmid pET28C-rPAL-1-cDNA with rice phenylalanine ammonia lyase(PAL) cDNA insert was constructed and its transformed E.coli BL21DE3 pET28C-rPAL-1-cDNA was established previously. Expression of the insert gene after isopropyl-β-D-thiogalactoside induction was demonstrated by SDS-PAGE and Mini-Protein two dimension electrophoresis. The molecular weight of the expressed fusion protein was estimated to be 78.6 ku, and was present mainly in the pellet(as the form of inclusion body) of the sonic disruptive bacteria. The specific activity of the pellet protein was 462 nmol·g -1·min -1 as measured by Zucker method and the K m of 0.50 mmol·L -1 and v max of 3.05 nmol·min -1 of the enzyme protein also estimated. Up to now, it was proved that the established E.coli BL21DE3 pET28C-rPAL-1-cDNA strain do express active PAL efficiently.