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目的建立重组靶向性抗肿瘤融合蛋白EGF-E4orf4毕赤酵母工程菌的中试发酵工艺。方法优化EGF-E4orf4工程菌二级种子培养时间,按5%的比例接种于20 L发酵培养基,设定培养阶段温度为30℃,诱导表达阶段温度为28℃,在发酵过程中通过调节搅拌速度、通气量和罐压等措施使DO维持在20%～35%,采用三步发酵法进行发酵,优化诱导pH值及诱导时间,并在已优化的工艺条件下,稳定发酵3批。结果确定二级种子的培养时间为15 h,最适诱导pH值为6.0,最适诱导时间为48 h;中试发酵3批,菌体A600均值达(327.67±17.96),菌体湿重均值达(308.33±9.07)g/L,EGF-E4orf4表达量均值为(200.00±5.57)mg/L。结论已初步建立了重组EGF-E4orf4毕赤酵母工程菌的中试发酵工艺,为EGF-E4orf4的产业化奠定了基础。
Objective To establish a pilot-scale fermentation process of recombinant targeted anti-tumor fusion protein EGF-E4orf4 Pichia pastoris. Methods The secondary seed culture time of EGF-E4orf4 engineering bacteria was optimized and inoculated into 20 L fermentation medium at the ratio of 5%. The temperature of culture stage was set at 30 ℃ and the induction stage was at 28 ℃. During the fermentation, Speed, aeration and tank pressure so that the DO maintained at 20% to 35%, three-step fermentation of fermentation, optimization of induced pH and induction time, and has been optimized in the fermentation process, stable fermentation three batches. The results showed that the second seed culture time was 15 h, the optimal pH value was 6.0 and the optimal induction time was 48 h. The average fermentation time was 48 h in three batches of trial fermentation, the average value of A600 was (327.67 ± 17.96) (308.33 ± 9.07) g / L, and the mean EGF-E4orf4 expression was (200.00 ± 5.57) mg / L. Conclusion The pilot fermentation process of recombinant Pichia pastoris EGF-E4orf4 has been preliminarily established, which laid the foundation for the industrialization of EGF-E4orf4.