巴斯德毕赤酵母表达人微小纤溶酶原的放大研究

Scale-up study of the Pichia pastoris expression system for human microplasminogen

目的:研究巴斯德毕赤酵母(Pichia pastoris)表达重组人微小纤溶酶原(rh-mPlg)的放大工艺。方法:分别采用1 L摇瓶、7.5 L发酵罐对Pichia pastoris工程菌rh-MPLG/GS115进行高密度培养、甲醇诱导表达。摇瓶培液经2步纯化:SP-Seph-arose FF、Superdex 75;发酵罐培液经3步纯化:超滤、Sephacryl S-100、Q-Sepharose FF,活性组分透析后冷冻干燥。以组织型纤溶酶原激活剂(t-PA)激活rh-mPlg,纤维蛋白平板测定其纤维蛋白溶解活性,计算并比较2种制备方法所获rh-mPlg比活性及得率。结果:采用1 L摇瓶和7.5 L发酵罐发酵可分别获得34 mg.L-1培液、210 mg.L-1培液的得率,经纯化后rh-mPlg纯度分别为95%和97%,比活性分别为20.6和23.0 U.mg-1。结论:以发酵罐生产rh-mPlg,可显著提高其产量,且比活性与摇瓶表达相近,验证了放大生产的可行性。

Objective：To study the scale-up methodologies of Pichia pastoris for expressing recombinant human microplasminogen（rh-mPlg）.Methods：1 L shake flasks and a 7.5 L fermenter was used for high density culture of engineered Pichia pastoris rh-MPLG/GS115 respectively.Methanol induction was performed to express rh-mPlg.The culture broth from 1 L shake flask was treated using a two-stage process：SP-Sepharose FF and Superdex 75;the broth from 7.5 L fermenter was treated using a three-stage processes：ultrafiltration,Sephacryl S-100 and Q-Sepharose FF.The active fraction was dialyzed and lyophilized.The rh-mPlg was activated by tissue plasminogen activator（t-PA）and its fibrinolytic activity was measured by fibrin plate method.The specific activities and recovery rates of the rh-mPlg by two preparative methods were calculated and compared respectively.Results：The preparation by 1 L shake flask and by 7.5 L fermenter yielded recovery rate of 34 mg rh-mPlg per liter fermentation broth and 210 mg per liter fermentation broth respectively.Through purification procedures,the purity of rh-mPlg of two preparations was 95% and 97% respectively;their specific activities were 20.6 U·mg-1 and 23.0 U·mg-1 respectively.Conclusion：The preparation by fermenters can prominently increase the rh-mPlg yields.The specific activity of intermediate production was comparable to that produced through shake flask,verifying the validation of scale-up production.