基因工程菌发酵表达rhG-CSF的条件优化

目的通过优化基因工程菌Escherichia coli BL21（DE3）Plys表达重组人粒细胞集落刺激因子（rhGCSF）蛋白的发酵工艺,提高蛋白产量。方法以100 L规模发酵为例,从不同的接种量（1%～10%）、加入IPTG进行诱导时的不同OD600nm值,诱导表达后的不同溶氧可控制范围（20%～80%）等方面进行优化。用SDS-PAGE对蛋白表达量进行检测。结果优化后的方案为：采用5%的接种量,在OD600nm达到10～15时加入诱导剂IPTG,诱导起始后前20 min控制溶氧在80%以下,20 min后通过补加氨水控制pH在7.0左右,补料调节控制溶氧在20%～40%的范围内,诱导表达5 h后收集菌体。优化后的发酵方案极大提高了蛋白表达量。结论 rhG-CSF蛋白发酵方案的优化对大规模生产具有重要的指导意义。

Optimization of the fermentation of rhG-CSF using recombined strain

Objective To improve the rhG-CSF protein from the recombined strain Escherichia coli BL21（DE3）Plys,the batch fermentation conditions was optimesed.Methods We determined the influence of the parameters at some crucial control points,including different inoculation amount（1% ～ 10%）,the OD600nmof fermentation culture when adding inducer IPTG and the different concentrations of dissolved oxygen（DO）（20% ～ 80%） on the final protein expression in100 L batch level fermentation.A SDS-PAGE was used to analyze the protein expression.Results The optimized fermentation condition was as follow：inoculum quantity 5%,IPTG should be added when the OD600nmof culture reaches 10 ～ 15,at the beginning of inducing the expression of object protein,the DO concentration was controlled under 80%; After inducing20 min,the pH of medium was kept at around 7.0 by adding ammonia,the DO concentration was kept at 20% ～ 40%; The bacteria was collected after inducing 5 hours.The production of rhG-CSF was higher after optimizing the fermentation conditions.Conclusion The optimized fermentation conditions for rhG-CSF production in E.coli BL21（DE3） Plys provided import direction for its large scale preparation.