天然N-末端人白细胞介素-29成熟肽在毕赤酵母中表达条件的优化

目的优化天然N-末端人白细胞介素-29(Human interleukin-29,hIL-29)成熟肽在毕赤酵母中的表达条件,高效表达天然N-末端hIL-29成熟肽。方法采用单因素试验和L(934)正交试验,分析摇瓶发酵条件下甲醇添加量、起始pH值、诱导时间及诱导温度对毕赤酵母工程菌GS115/hIL-29发酵液A450值的影响;用5 L发酵罐初步探索工程菌株的高密度发酵策略。结果影响重组毕赤酵母摇瓶发酵液A450值的因素重要性依次为:诱导温度>起始pH值>甲醇添加量>诱导时间,最佳发酵条件为:起始pH值7.5、甲醇添加量1.5%、26℃诱导60 h,在此条件下,发酵液的A450值达1.72;高密度发酵策略为:pH 7.5,甲醇与溶氧反相偶联,26℃诱导12 h,在此条件下,目的蛋白的表达量明显高于摇瓶水平;高密度发酵12、24 h表达的蛋白与羊抗人IL-29多抗的反应强度明显高于摇瓶发酵表达的蛋白。结论优化了工程菌株GS115/hIL-29摇瓶发酵和发酵罐高密度发酵条件,实现了重组天然N-末端hIL-29成熟肽在毕赤酵母GS115中的高效表达,为其进一步的中试放大工艺奠定了基础。

Optimization of condition for expression of native N-terminus of human interleukin-29 mature peptide in Pichia pastoris

Objective To optimize the condition for expression of native N-terminus of human interleukin-29(IL-29) mature peptide in Pichia pastoris.Methods The effects of culture conditions such as methanol concentration,initial pH value of medium as well as time and temperature for induction on the A450 value of recombinant P.pastoris GS115 / hIL-29 fermented in shake flask were investigated by using single factor test and L(934)orthogonal test,based on which the strategy for high density fermentation of recombinant P.pastoris in 5 L fermenter was preliminary explored.Results In the turn of importance,the influencing factors of A450 value of fermentation liquid in shake flask were induction temperature,initial pH value of medium,methanol concentration and induction time.The optimal initial pH value of medium,methanol concentration,temperature and time for induction were 7.5,1.5%,26℃ and 60 h respectively.Under the optimized condition,the A450 value of fermentation liquid reached 1.72.The strategy for high density fermentation strategy was as follows:pH 7.5,reverse phase coupling of methanol and dissolved oxygen,and induction at 26℃ for 12 h.The expression level of target protein by high density fermentation was significantly higher than that by fermentation in shake flask.However,the reaction strengths of expressed protein in P.pastoris 12 and 24 h after high density fermentation with goat anti-human polyclonal antibody against IL-29 were significantly higher than those after fermentation in shake flask.Conclusion The conditions for fermentation in shake flask and high density fermentation in fermenter of recombinant P.pastoris GS115 / hIL-29 were optimized,and native N-terminus of human IL-29 mature peptide was highly ex pressed,which laid a foundation of further scale-up of pilot process.