人胰激肽原酶在毕赤酵母中的分泌表达

人胰激肽原酶(hPK)编码基因由本实验室设计、合成,并重组构建成表达质粒pPIC9k-hPK。经电穿孔将该质粒转化毕赤酵母GS115 (His^-Mut⁺) ,筛选His⁺Mut^s 型高拷贝菌株,摇瓶诱导表达重组hPK。经SDS-PAGE凝胶电泳分析确定表达重组蛋白相对分子质量为37500,产量达40mg/L,质量分数占总蛋白的30%以上。初步浓缩的重组hPK经测定具有酶活性,表明其在毕赤酵母中得到了正确的表达,每mL酶活达0.717nmol/s。

Secretory expression of human pancreatic kallikrein in Pichia pastoris

Pancreatic kallikrein (PK) mentioned in the article has remarkable effect on the treatment of hypertension and thrombosis. Human pancreatic kallikrein (hPK) possesses a potential commercial value due to its characterization of higher effective and more compatible than PK from animal tissue. In the study, a DNA fragment encoding hPK was synthesized and inserted into the plasmid pPIC9K to construct the expression vector pPIC9K-hPK and following transformed the host cell yeast Pichia pastoris GS115 by means of elctroporation. By a serial screening His Mut s positive clones and multi-copy transformants were identified. One of clones was cultured in flask for the expression of human pancreatic kallikrein in vitro. The band of molecular weight about 37500 was observed on SDS-PAGE, and the recombinant hPK was about 30% of total soluble proteins by gel analysis.The yield of fully active enzyme was about 40mg/L. After concentration by ultrafiltration, it was proved that hPK was exactly expressed in Pichia pastoris and the enzyme activity reached 0.717nmol/s per ml.