发酵条件对毕赤酵母表达重组人干扰素ω糖基化的影响

The Effect of Fermentation Conditions on Glycosylation of Recombinant Human Interferon Omega in Yeast Pichia pastoris

发酵条件是影响毕赤酵母 (P .pastoris)表达外源重组糖蛋白时糖基化的重要因素。通过菌体浓度、起始pH值、甲醇诱导浓度和周期、装液量等摇瓶发酵实验 ,研究不同发酵条件对毕赤酵母表达分泌型重组人干扰素ω(rhIFNω)过程中糖基化的影响 ;同时 ,在连续培养过程中考察pH值变化对rhIFNω糖基化的影响和分批发酵过程中rhIFNω糖基化的变化。结果表明 ,控制菌体密度 2 5 0g L(WCW)、起始pH值 6 0、装液量小于 30mL、甲醇诱导浓度 15g L、甲醇诱导 3次 (每 2 4h诱导一次 )等发酵条件 ,有利于摇瓶发酵过程中rhIFNω的糖基化 ;控制pH值 7 0～ 7 5可促进rhIFNω的糖基化 ;分批发酵过程中 ,糖基化与非糖基化rhIFNω的含量有同比变化趋势 ,但糖基化rhIFNω所占比例明显低于摇瓶发酵实验的结果 ,其原因有待进一步研究。

To investigate the influence of the fermentation conditions on glycosylation of heterologous recombinant protein in yeast Pichia pastoris , the glycosylation of recombinant human interferon omega(rhIFNω) under various fermentation conditions, e.g., cell density, initial pH, methanol concentration, duration of the induction, and medium volume were studied. The glycosylation of rhIFNω in the continuous fermentation process under various pH values and in batch fermentation were also investigated. In 250 mL flask, the optimal cell density, initial pH, medium volume, methanol concentration and frequency of methanol induction were 250 g/L(WCW), pH6 0, less than 30 mL, 15 g/L and 3 (in every 24 h), respectively. In the continuous process, the glycosylation of rhIFNω could be effectively improved by maintaining the pH value at 7 0～7 5 In the batch fermentation process, the expression level of glycosylated and non_glycosylated rhIFNω were the same, but the specified value of glycosylation/non_glycosylation was significantly lower than that in the flask culture. The reason of this phenomenon will be further studied. This research lay the foundation for the scale_up of production and the enhancement of rhIFNω glycosylation in Pichia pastoris .