大肠杆菌是用于产生抗体和抗体片段的最合适的宿主之一。抗体片段分泌到培养基中提高了细胞培养物中的产物纯度并降低了下游成本。在这项研究中,选择携带编码双特异性抗MUC1纳米抗体的基因的大肠杆菌菌株BL21(DE3),并研究了双特异性抗MUC1纳米抗体表达的自诱导方法。由于用乳糖作为诱导剂取代IPTG,在最终产品中观察到较少的杂质和毒性。为了增加细胞内和细胞外纳米抗体的产生,最初,这些实验是针对包括温度和蛋白质表达持续时间在内的关键因素进行的。在33°C下21小时后产生最高量的纳米体。不同碳源的影响,甘油,葡萄糖,乳糖,和甘氨酸作为介质添加剂在最佳温度和时间也通过使用响应面法进行了评估。最佳碳源浓度为0.75%(w/v),0.03%(w/v),0.1%(w/v),甘油为0.75%(w/v),葡萄糖,乳糖,和甘氨酸,分别。最后,评估了在优化的自诱导条件下2L发酵罐中纳米抗体的产生。结果表明,总滴度为87.66µg/mL的抗MUC1纳米抗体,大约是LB培养基中产生的纳米抗体总滴度的七倍,为12.23微克/升。
Escherichia coli is one of the most suitable hosts for production of antibodies and antibody fragments. Antibody fragment secretion to the culture medium improves product purity in cell culture and diminishes downstream costs. In this study, E. coli strain BL21 (DE3) harboring gene encoding bispecific anti-MUC1 nanobody was selected, and the autoinduction methodology for expression of bispecific anti-MUC1 nanobody was investigated. Due to the replacement of IPTG by lactose as inducer, less impurity and toxicity in the final product were observed. To increase both intracellular and extracellular nanobody production, initially, the experiments were performed for the key factors including temperature and duration of protein expression. The highest amount of nanobody was produced after 21 h at 33°C. The effect of different carbon sources, glycerol, glucose, lactose, and glycine as a medium additive at optimum temperature and time were also assessed by using response surface methodology. The optimized concentrations of carbon sources were obtained as 0.75% (w/v), 0.03% (w/v), 0.1% (w/v), and 0.75% (w/v) for glycerol, glucose, lactose, and glycine, respectively. Finally, the production of nanobody in 2 L fermenter under the optimized autoinduction conditions was evaluated. The results show that the total titer of 87.66 µg/mL anti-MUC1 nanobody, which is approximately seven times more than the total titer of nanobody produced in LB culture medium, is 12.23 µg/L .