【摘要】 采用三角摇瓶来摸索BL21(DE3)/pET28A-Tα1菌种表达目的蛋白的最优发酵条件,并在此基础上在Biotop CF-5L自动控制发酵罐中,利用补料分批培养技术,流加甘油,进行发酵培养;利用Western blotting进行发酵产物鉴定.摇瓶培养最优条件:10%接种量、25 mL LB培养基、37℃培养,0.02 g/mL的甘油做碳源,接种后4 h加入终浓度为0.75 mmol/L的IPTG,诱导3h后目的蛋白表达量最高.BL21(DE3)/pET28A-Tα1菌种在BiotopCF-5L自动控制发酵罐中的条件为:以10%的接种量接种到3L发酵培养基中,设定溶氧40%,pH7.0,温度37℃,通气量3 L/min,快速流加甘油60 g,培养4 h后,加入终浓度为0.75 mmol/L的IPTG,诱导3 h后终止发酵.发酵罐中获得的菌体量为36 g/L,蛋白表达率为10%左右.更多还原

【Abstract】 To express Tα1 in BL21/pET28a-Tα1,the conditions for gene expression was optimized in a trail cell culture in flask followed by culturing in Biotop CF-5L fermenter using fed-batch fermentation.The expression of the target gene was verified by western blot assay.It was found that the best expression could be achieved by growing of the cells,started from 10% inoculation,in LB medium with 0.02 g/mL Glycerol as a carbon source for 4 hours,followed by additional 4 hrs induction with 0.75 mmol/L IPTG.Large scale gene expression was performed in a Biotop CF-5L fermenter.Started with 10% inoculation,the cells were cultured in 3 L pH 7 fermentation medium supplemented with 40% DO and Glycerol feed at 37 ℃ for 4 hours,followed by 3 hrs induction with 0.75 mmol/L IPTG.The final biomass was 36 g/L and the expressed recombinant Tα1 was about 10% of total cellular protein.更多还原