High-cell-density Cultivation of Escherichia coli Expressing Human Keratinocyte Growth Factor
- 化学化工－已发表论文 
[中文摘要]角质细胞生长因子(KGF)是成纤维生长因子家族(FGF)成员,与上皮细胞增殖、组织胚胎发育、创伤修复等过程密切相关.在大肠杆菌BL21(DE3)中克隆KGF基因,并进行诱导表达条件的优化.使用合成培养基,应用底物反馈流加策略将糖浓度控制在较低水平,在发酵罐中高密度培养,细胞干质量浓度可以达到46 g/L.发酵罐培养的大肠杆菌在对数生长期经过异丙基-β-D-硫代半乳糖苷(IPTG)诱导,在可溶性和不溶性产物中都得到了具有免疫学活性的GST-KGF融合蛋白,经过ELISA检测,发酵液中可溶性表达的KGF产率达到108μg/L.该研究为利用原核表达系统生产活性KGF建立了优化工艺,有利于将大规模生产用于创伤修复的KGF.[英文摘要]Keratinocyte growth factor(KGF) is the seventh member of the fibroblast growth factor family,and plays important role in the process of the proliferation of epithelial cell,the development of embryonal tissue and wound repairing.In this study,the Escherichia coli stain BL21(DE3) cloned with the plasmid pGEX-2T-KGF to expressing the fusion protein GST-KGF.The optimized conditions of induction temperature,the density of bacteria,induction time and inducer concentration for expression of fusion protein GST-KGF were investigated. When cultivating in Riesenberg synthetic medium without isopropyl β-D-thiog alactopy ranoside (IPTG) induction, feedback control of substrate was applied, and the concerntr ation of glucose was controlled between 0.2g/L and 0.5 g/L, the cell dry mass concentration could reach 46g/L. Cultiv ating in ferment or and intro duced by IPTG in the exponential phase, the immunological activated fusion protein GST-KGF from both soluble and insoluble product were obtained by ELISA determination,108 μg/L KGF from soluble product could be achieved. An optimized process is established for production of activated human KGF by prokaryotic expression system, which lays a foundation for mass production of KGF for wound healing .