摘要
目的:优化融合表达抑肽酶基因工程菌的发酵条件,分离纯化抑肽酶。方法:设计3因素3水平的正交实验,考查玉米粉浓度、诱导时间和诱导剂乳糖浓度对菌体生长及蛋白表达的影响;在30 L发酵罐中进行中试放大试验;菌体经超声破壁和离心得到融合蛋白包涵体,经Bio-gel P30凝胶过滤纯化复性,用FXa切割,回收目的蛋白,最后用Resource RPC进行纯度验证。结果:融合蛋白的表达量为45%,包涵体复性率为70%以上,抑肽酶纯度为90%,总收率为32%,比活力为3.1 EPU/mg。结论:实现了含抑肽酶融合蛋白的较高表达,复性率及纯度达到较高水平,活力与天然产物相当。
Aim： To optimize the fermentation conditions of engineering bacteria expressing fusion proteins composed of aprotinin and to purify aprotinin. Methods： Orthogonal design was used to examine the influences of corn powder concentration, inducing time and the concentration of lactose as an inductor on the growth of cells and the expression level. 30 L fermentor was applied to carry out the scale-up production. Cells were collected by centrifugation and broke through by ultrasound. After renaturation and purification by Bio-gel P30, the fusion protein was then specifically digested with FXa and the recombinant aprotinin was obtained, which was confirmed to have the same characteristics as native aprotinin. Its purity was further analyzed by Resource RPC. Results：The fusion protein level, renaturation of inclusion bodies, purity of aprotinin, total recovery and the activity were about 45%, 70%, 90%, 32% and 3.1 EPU/mg respectively. Conclusion： High level expression of the fusion protein composed of aprotinin was achieved with satisfactory renaturation rate and purity and the equivalent activity with the natural aprotinin was obtained.
出处
《中国药科大学学报》
CAS
CSCD
北大核心
2006年第4期375-378,共4页
Journal of China Pharmaceutical University
