猪IFNα在毕赤酵母中分泌表达条件的优化及高密度发酵

Studies on Expression Conditions of PoIFNα and High-density Fermentation for Genetically Engineered Pichia Pastoris

从发酵时间、接种量、pH值、诱导剂量等方面对重组基因工程(毕赤酵母甲醇利用缓慢型)的摇瓶发酵条件进行了优化,进一步探索了酵母菌表达猪IFNα的发酵工艺,包括种细胞密度对初始发酵期的影响,补加甘油、甲醇速率条件的控制等.结果表明,摇瓶发酵时,诱导最佳时间为96 h,甲醇最适浓度为8 g/L,发酵pH范围为6.4~9.0,最适接种量2∶1.在分批发酵、接种量为10%且种子细胞光密度(OD600)为5~6时,最有利于细胞的高密度培养,在补料发酵时,根据溶氧控制甘油流加速率与时机,细胞干重达到118.75 g/L,在48 h重组猪IFNα表达量达到1.304 g/L.

The optimum fermentation conditions of genetically engineered Pichia pastoris in shake-flask cultivation and in fed-batch fermentation are reported respectively in this paper. Experimental results revealed that the fermentation period induced with methanol is 96 h. The optimum methanol is 8 g/L, pH range is 6.4 - 9.0 and the seed inoeulum amount is 2 ： 1 in shake-flask fermentation. Although the seed inoeulum amount increases, the target protein concentration decreases. The 10% inoeulum and 5 - 6 OD^oo of seed are benefit to the high density cultivation. With glycerol feeding strategy based on DO level, the cell dry weight of broth reaches 118.7 g/L and the IFN concentration is 1. 034 g/L by methanol inducement for 48 hours at the fed-batch fermentation phase.