| 巴斯德毕赤酵母表达人微小纤溶酶原的放大研究 |
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| 引用本文： | 陈武,莫炜,张艳玲,宋后燕. 巴斯德毕赤酵母表达人微小纤溶酶原的放大研究[J]. 药物分析杂志, 2011, 0(1) |
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| 作者姓名： | 陈武 莫炜 张艳玲 宋后燕 |
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| 作者单位： | 广东药学院生命科学与生物制药学院;复旦大学分子医学教育部重点实验室; |
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| 基金项目： | 广东药学院博士基金项目(43555026) |
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| 摘 要： | 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。
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| 关 键 词： | 巴斯德毕赤酵母 纤溶酶原 表达 纯化 放大 |
Scale-up study of the Pichia pastoris expression system for human microplasminogen |
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| CHEN Wu,,MO Wei,ZHANG Yan-ling,SONG Hou-yan. Scale-up study of the Pichia pastoris expression system for human microplasminogen[J]. Chinese Journal of Pharmaceutical Analysis, 2011, 0(1) |
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| Authors: | CHEN Wu MO Wei ZHANG Yan-ling SONG Hou-yan |
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| Affiliation: | CHEN Wu1,2,MO Wei2,ZHANG Yan-ling2,SONG Hou-yan2(1.The Life Science and Biopharmacy College,Guangdong Pharmaceutics University,Guangzhou 510006,China,2.The Key Laboratory of Molecular Medicine,Ministry of Education,Fudan University,Shanghai 200032,China) |
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| Abstract: | 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,... |
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| Keywords: | Pichia pastoris plasminogen expression purification scale-up |
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