中华鲟cystatin酵母工程菌的发酵条件和表达产物的纯化

Fermentative Optimization and Purification of Yeast Recombinant Acipenser sinensis Cystatin

为进一步开展重组中华鲟(Acipenser sinensis)半胱氨酸蛋白酶抑制剂cystatin功能分析和应用研究,进行了诱导方式、诱导时间和培养基pH值对该重组菌的表达量的影响研究。结果表明,用直接诱导法,在诱导72 h、pH值为6.5、甲醇终浓度为0.5％的条件下,对重组酵母进行诱导最为合适,重组cystatin表达量可达每升培养基215 mg,约占培养基上清总蛋白量的76.3％。经离子交换层析法纯化,重组cystatin的纯度可达94.2％。生物活性检测试验表明。该方法提取的重组cystatin对木瓜蛋白酶的活性具有明显的抑制作用。

Cystatin is an inhibitor of cysteine proteinases. In order to apply the recombinant Aci-penser sinensis cystatin to fish disease prevention and cure, as well as seafood process industry, the expression levels with three inductive methods, at different inductive time points and pH values, were tested. Results indicated that after 72 h induction, the highest expression level was obtained at pH6. 5 in only one medium (BMGY). Density scanning of the SDS-PAGE gel revealed that the expressed protein accounted for about 73. 6% in the culture supernatant, and the expression level is about 215 mg per liter medium. After purified through Q-Sepharose anion-exchange chromatography the purity of the recombinant cystatin was about 94. 2%. The inhibitory activity of recombinant cystatin was measured by inhibiting the protease activity of papain.