可溶性TRAIL蛋白的高密度培养及补料策略研究  

High-cell Density Cultivation of Recombinant Escherichia coli for Production of TRAIL by Using a 2-stage Feeding Strategy

作　　者：章越[1] 沈亚领[1] 夏小霞[1] 孙爱友[1] 魏东芝[1] 周劲松 张国钧 王梁华[3] 焦炳华[3] 

机构地区：[1]华东理工大学生物化学研究所,生物反应器工程国家重点实验室,上海200237 [2]上海恰尔生物技术有限公司,上海200336 [3]第二军医大学基础医学部生物化学与分子生物学教研室,上海200433

出　　处：《生物工程学报》2004年第3期408-413,共6页Chinese Journal of Biotechnology

基　　金：国家重大科技专项基金资助 (No .2 0 0 2AA2Z3 45A)~~

摘　　要：采用分批补料的方法高密度培养重组大肠杆菌C6 0 0 pBV TRAIL制备人可溶性TRAIL蛋白 ,优化发酵工艺 ,探索简单高效的分离纯化方法并测定蛋白生物活性。通过比较几种不同的补料策略 :间歇流加、DO stat、pH stat,摸索了一种流加策略 ,即DO stat pH stat组合流加 ,有效的避免了发酵过程中 ,尤其是诱导表达阶段乙酸积累的增加 ,使TRAIL蛋白在高密度培养条件下 ,得到高效表达。菌体密度最终达到 30 0g L(WCW )以上 ,可溶性TRAIL蛋白占菌体总蛋白的 4 2 % ,含量为 1 1g L。在整个发酵过程中 ,乙酸浓度接近于 0 ,且未使用任何特殊手段 ,如纯氧、加压等 ,简化了发酵工艺 ,降低了发酵成本 ,为TRAIL的工业化生产创造了条件。Escherichia coli was genetically engineered to produce recombinant tumor necrosis factor-related apoptosis inducing ligand (Apo2L/TRAIL) using a temperature-inducible expression system. To create a fed-batch culture condition that allows efficient production of TRAIL, different feeding strategy including discontinuous, DO-stat and pH-stat feeding strategies were compared. Then, a special 2-stage feeding strategy was developed. High concentration of biomass (300g wet cell weight per liter of culture broth) and active soluble TRAIL protein (1.1g/L) was obtained by applying a high-cell-density cultivation procedure with the 2-stage feeding strategy. Cultivation of recombinant E.coli was started as a batch process at 30℃ and then followed by fed-batch culture when the dissolved oxygen concentration presented a steep increase resulted from the exhaustion of glucose in the medium. At the first phase of fermentation (batch phase), agitation rate was enhanced to control dissolved oxygen at 30 percent. When glucose in the medium was used up, indicated by a sudden rise in pH value and dissolved oxygen, the second phase (fed-batch phase) was started with glucose and nitrogen resource being supplied automatically. At the beginning of fed-batch operation, stirrer rate was cascaded with dissolved oxygen signals to keep it at 20 percent(DO-stat).During the fed-batch phase, glucose was limited to control the specific growth rate under the critical value μcrit,to avoid acetic acid excretion. When the stirrer speed arrived at its up-limit, the flow rate of feed was kept constant. In the inducing phase(42℃ for 4h) glucose was fed as a pH regulating agent(pH-stat) and the specific growth rate and dissolved oxygen decreased sharply. Aqueous ammonia was used for maintaining pH value at 7.0 throughout the first two phases. In the whole fermentation, acetic acid concentration didn't exceed 2.9 g/L. At the end of the high-cell-density cultivation process, no acetic acid could be detected in the medium. These results indicated that

关 键 词：重组大肠杆菌 分批补料 发酵 TRAIL 分离纯化 

分 类 号：Q819[生物学—生物工程]