【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。更多还原

【Abstract】 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 our fed-batch strategy was able to prevent acetate accumulation significantly. Although high cell density has been achieved, the induction process was not optimized satisfactorily and much work should be done further. Furthermore, since no special ways, like pure oxygen, pressure, has been used in our experiments, this efficient approaches would be useful not only in a pilot scale but also in an industry scale.;Finally, simple purification procedure based on immobilized metal affinity column (IMAC) and CM-Sepharose column was implemented to isolate the TRAIL. Yields of more than 800mg TRAIL per liter of culture broth were obtained, the final purity reaching more than 95%. The purified TRAIL showed strong cytotoxity activity against human pancreatic 1990 tumor cells, with ED 50 about 1.6 μg/mL.更多还原