人乙醛脱氢酶2基因在毕赤酵母中的高效表达

Overexpression of Human Acetaldehyde Dehydrogenase 2 in Pichia Pastoris

去除自行构建的质粒pPIC9K-07ALDH2的信号肽(α-Factor)得到质粒pPIC9K-ALDH2-delsign,采用电转化方法将该质粒转化到Pichia pastorisSMD1168中构建得到能在胞内高效表达人乙醛脱氢酶2(ALDH2)的基因工程菌株Pichia pastorisSMD1168(pPIC9K-ALDH2-delsign)。利用该重组毕赤酵母摇瓶发酵得到的发酵液的人ALDH2的酶活为0.315 U.mL-1,明显高于胞外表达时人ALDH2的酶活;经过亲和色谱分离纯化后的人ALDH2酶液的酶活为0.944 U.mL-1。

In this study a new plasmid named pPIC9K-ALDH2-delsign by removing the a-factor of plasmid pPIC9K-07ALDH2 was gained. The pPIC9K-ALDH2-delsign was linearized by SacI and transformed by electroporation into Pichia pastoris SMD1168. Pichia pastoris SMD1168 （pPICgK-ALDH2-delsign） was able to overexpress human ALDH2 within the cell. The activity of human ALDH2 of cultured supernatant fermented by shaking flask was approximately 0. 315 U · mL^-1 , which was obviously higher than that in extracellular expression. The activity of human ALDH2 purified by affinity chromatography was 0. 944 U · mL^-1.