Optimization for Purification and Characterization of Recombinant Hirudin Ⅲ from E. coli  

分离纯化重组水蛭素Ⅲ工艺的优化研究及重组水蛭素Ⅲ的分子鉴定(英文)

作　　者：韦利军[1] 刘军 吴斌 李雪峰[1] 叶双宁[1] 章良[1] 吴梧桐[1] 

机构地区：[1]中国药科大学生命科学与技术学院,江苏南京210009 [2]常州千红生化制药公司,江苏常州213000

出　　处：《Journal of Chinese Pharmaceutical Sciences》2005年第2期79-85,共7页中国药学（英文版）

摘　　要：Aim To optimize purification conditions of recombinant hirudin 3 in thefermentation broth and characterize the product. Methods Reambinant hirudin 3 was isolated andpurified from the fermentation broth by three column chromatography steps with macroporous resin,DEAE cellulose DES2 and preparative RP-HPLC, respectively, and the optimal conditions were obtained.Purity of the product was determined by SDS-PAGE and analytical RP-HPLC. The molecular weight wasdetermined by mass spec-trometry. The structure of the product was analyzed by peptide map.ResultsThe product with purity of 95.4786% was obtained after three purification steps in the optimumconditions with a total yield of 39%. The molecular weight of the product was 6 913.32 ± 6.55 Da,coincident to the theoretical molecular weight of r-hirudin 3. The structure of the product wascoincident to r-hirudin 3 either. Conclusion The optimized purification steps can be successfullyemployed for purification of r-hirudin 3 from E. coli using batch-type approaches. The productobtained with high purity was confirmed to be r-hirudin 3.目的优化分离纯化大肠杆菌发酵液中重组水蛭素Ⅲ的工艺并对所纯化产物进行分子鉴定。方法通过三步纯化步骤,即大孔树脂层析、DEAE纤维素DE52层析和反相高效液相色谱层析,分离纯化发酵液中的重组水蛭素Ⅲ,优化纯化条件。通过SDSPAGE和分析型反相高效液相色谱对重组水蛭素Ⅲ的纯度进行鉴定。以质谱对重组水蛭素Ⅲ的分子量进行鉴定。通过肽图的测定来鉴定重组水蛭素Ⅲ的结构。结果通过优化后的分离纯化步骤得到了纯度为95.4786%的纯品,总产率为39%。得到的产物分子量为6913.32±6.55Da,与重组水蛭素Ⅲ分子量的理论值相符,其结构亦与理论值一致。结论优化后的分离纯化步骤可以有效地用于大肠杆菌发酵液中重组水蛭素Ⅲ的大规模分离纯化。所得的高纯度产物确为基因重组水蛭素Ⅲ。

关 键 词：recombinant hirudin 3 PURIFICATION macroporous resin RP-HPLC massspeetrome- try peptide map 

分 类 号：TQ464[化学工程—制药化工]