表达重组人可溶性TRAIL的毕氏酵母发酵工艺

目的优化rhsTRAIL毕氏酵母工程菌在5L发酵罐中的发酵表达工艺参数。方法研究培养基种类、细胞密度、甘油含量、pH值、甲醇浓度、甲醇流加速率及诱导时间等参数对工程菌生长及目的蛋白表达的影响,并用电镜观察其诱导肿瘤细胞凋亡特征。结果在无机盐培养基中,按10%接种A600=8～12的种菌后,24h酵母菌增殖至A600=76;甘油含量为1%时,菌体生长速度快(A600=160);培养基pH值5.0,甲醇终浓度为1%时,诱导96h表达量最高达到120mg/L,并具有诱导肿瘤细胞凋亡特征。结论rhsTRAIL毕氏酵母的发酵工艺参数为无机盐培养基,pH值5.0,接种10%A600=8～12的种菌,甘油含量1%,甲醇终浓度1%,诱导96h。

Fermentation Process of Pichia pastoris Expressing Recombinant Human Soluble TRAIL

Objective To optimize the fermentation process of Pichia pastoris expressing recombinant human soluble TRAIL(rhsTRAIL) in a 5 L fermentor.Methods Study the influence of fermentation parameters,such as medium,seed cell density,glycerol content,pH value,the concentration and flow rate of methanol,as well as the time for induction,on the growth of Pichia pastoris and expression of target protein.Determine the activity of rhTRAIL in inducing the aoptosis of tumor cells by electron microscopy.Results After inorganic salt medium was inoculated with the seed cells at a density(A_ 600 ) of 8-12 and a m.o.i of 10% and incubated for 24 hours,the density of Pichia pastoris(A_ 600 ) reached 76.When the glycerol content was 1%,the Pichia pastoris grew rapidly(A_ 600 =160).When Pichia pastoris was incubated in the medium at a pH value of 5.0 and a final methanol concentration of 1% for 96 hours,the rhsTRAIL reached a maximum expression level of 120 mg/L,and showed the activity in inducing the apoptosis of tumor cells.Conclusion The fermentation process of Pichia pastoris expressing rhsTRAIL was optimized as follows:To inorganic salt medium(pH 5.0),inoculate the seed cells of Pichia pastoris at a density(A_ 600 ) of 8-12 and a m.o.i of 10%,add glycerol and methanol to a final concentration of 1% separately and induce for 96 hours.