苯丙氨酸脱氨酶cDNA在大肠杆菌中的克隆与表达

Cloning and Expression of Phenylalanine Ammonia Lyase cDNA in Escherichia coli

利用逆转录聚合酶链反应（ＲＴＰＣＲ）技术从欧芹总ＲＮＡ分离制备了苯丙氨酸脱氨酶（ＰＡＬ）ｃＤＮＡ，重组到质粒ｐＢｌｕｅｓｃｒｉｐｔ及ｐＥＴ２３ｂ中，后者转化大肠杆菌ＪＭ１０９ＤＥ３获得ＰＡＬ的高效表达。插入片段两端共９２０ｂｐ的测序结果与文献符合率为９９７８％。工程菌细胞裂解液用ＳＤＳＰＡＧＥ（聚丙烯酰胺凝胶电泳）分析显示在分子量７７ｋＤａ处有一条表达很强的蛋白区带，与ＰＡＬ亚基分子量相符，约占菌体总蛋白的１５％。用ＨＰＬＣ法检测到苯丙氨酸被ＰＡＬ脱氨的产物肉桂酸，且该ＰＡＬ酶活力特异性好，适于ＰＫＵ治疗之用。

The phenylalanine ammonia lyase (PAL) cDNA was amplified from Petroselinum crispum total RNA by using RT PCR technique.The amplified 2.2kb DNA fragment was inserted into expression vecter pET23b.The resulting plasmid pET23bPAL was sequenced and then transformed into E.coli JM109DE3.The expressed PAL protein in JM109DE3(pET23bPAL)accounted for more than 15% of the total proteins in the recombinent E.coli cell.The activity and specificity of the expressed PAL was identified and tested by using HPLC technique,and showed good enough for application to enzymatic therapy of PKU.