重组人组织型纤溶酶原激活剂改构体的表达、复性与分离纯化  

Study on expression, renaturation and purification of reteplase

作　　者：冯长根[1] 陈嫚[1] 任启生[2] 宋新荣[2] 

机构地区：[1]北京理工大学新医药开发研究中心,北京100081 [2]北京江中泽生科技有限责任公司,北京100050

出　　处：《中国药学杂志》2004年第7期550-552,557,共4页Chinese Pharmaceutical Journal

摘　　要：目的 获得具有高纯度、高生物活性的重组人组织型纤溶酶原激活剂改构体(瑞替普酶,reteplase,r-PA)。方法 reteplase基因克隆到表达质粒pJZ-100中,转化Escherichia cli B121(DE3),在0.05 mmol·L-1异丙基硫代-β-D-半乳糖苷(IPTG)诱导下获得高效表达,表达产物通过脉冲稀释法复性,复性后的蛋白质用ETI-Sepharose 4B亲和色谱纯化。结果 表达产物占菌体总蛋白质的20％,脉冲稀释法复性后复性收率为28％,经纯化后纯度达96％以上,比活为580 000 IU·mg-1。结论 建立了reteplase表达、复性与纯化工艺,以此工艺可获得高活性高纯度的reteplase。OBJECTIVE: To obtain the recombinant human tissue-type plasminogen activator mutant(reteplase, r-PA) with high purity and high specific activity. METHODS: Reteplase sequence was cloned into pJZ-100 expression vector. Being induced by IPTG, reteplase was highly expressed in Escherichia coli BL21 (DE3). The activity of expressed protein was recovered by step-wise dilution in vitro. The reteplase was purified by affinity chromatography. RESULTS: Expressed reteplase constituted more than 20% of total bacterial protein. The activity recovery by step-wise dilution was 28%. After purification, the purity of reteplase was above 96% and its specific activity was 580 000 IU&middot mg -1. CONCLUSION: The method of reteplase expression, renaturation and purification was established. The process can be a scheme to obtain reteplase with high purity and the high activity from E. coli.

关 键 词：重组人组织型纤溶酶原激活剂改构体 表达 复性 纯化 

分 类 号：Q814[生物学—生物工程]