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

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

韦利军, 刘军, 吴斌, 李雪峰, 叶双宁, 章良, 吴梧桐^*

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

收稿日期:2004-10-18 修回日期:2005-05-10 出版日期:2005-06-15 发布日期:2005-06-15
通讯作者: 吴梧桐*

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

WEI Li-jun, LIU Jun, WU Bin, LI Xue-feng, YE Shuang-ning, ZHANG Liang, WU Wu-tong^*

1.School of Life Sciences and Technology, China Pharmaceutical University, Nanjing 210009, China;
2.Changzhou Qianhong Biopharma Co. Ltd, Changzhou 213000, China

Received:2004-10-18 Revised:2005-05-10 Online:2005-06-15 Published:2005-06-15
Contact: WU Wu-tong*

摘要/Abstract

摘要： 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: 重组水蛭素III, 重组水蛭素III, 重组水蛭素III, 纯化, 纯化, 纯化, 大孔树脂, 大孔树脂, 大孔树脂, 反相高效液相色谱, 反相高效液相色谱, 反相高效液相色谱, 质谱, 质谱, 质谱, 肽图, 肽图, 肽图

Abstract: Aim To optimize purification conditions of recombinant hirudin 3 in the fermentation broth and characterize the product. Methods Reambinant hirudin 3 was isolated and purified from the fermentation broth by three column chromatography steps with macroporous resin, DEAE cellulose DE52 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 was determined by mass spectrometry. The structure of the product was analyzed by peptide map. Results The product with purity of 95.4786% was obtained after three purification steps in the optimum conditions 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 was coincident to r-hirudin 3 either. Conclusion The optimized purification steps can be successfully employed for purification of r-hirudin 3 from E. coli using batch-type approaches. The product obtained with high purity was confirmed to be r-hirudin 3.

Key words: recombinant hirudin 3, recombinant hirudin 3, purification, purification, macroporous resin, macroporous resin, RP-HPLC, RP-HPLC, mass spectrometry, mass spectrometry, peptide map, peptide map

中图分类号:

R917.796

R996.3

Supporting: ^*Corresponding author. Tel.: 86-25-3220372; fax: 86-25-3220372

韦利军, 刘军, 吴斌, 李雪峰, 叶双宁, 章良, 吴梧桐^*. 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[J]. .

WEI Li-jun, LIU Jun, WU Bin, LI Xue-feng, YE Shuang-ning, ZHANG Liang, WU Wu-tong^*. Optimization for Purification and Characterization of Recombinant Hirudin III from E. coli[J]. .

参考文献

[1] Sohn JH, Kang HA, Rao KJ, et al. Current status of the anticoagulant hirudin : its biotechnological production and clinical practice [J]. Appl Microbiol Biotechnol, 2001, 57 : 606-613.
[2] Li TQ, Wan CX, LI YJ, et al. A novel high quality specific antithrombotic drug-hirudin [J]. J Biomed Eng, 1998, 15 (3) : 306-310.
[3] Chen JC, Wang YJ, Li J. Protein engineering of antithrombolic hirolog from leech-Thought on development of bioengineering of TCM in China [J]. Chin Tradit Herb Drugs, 2001, 32 (2) :178-179.
[4] Tan SH, Wu WT, Liu JJ, et al. Efficient expression and secretion of recombinant hirudin III in E. coli using the L-asparaginase II signal sequence [J]. Protein Expression Purification, 2002, 25 : 430-436.
[5] Fritz M. The development of hirudin as an antithrombotic drug [J]. Thromb Res, 1994,74 :1-23.
[6] Stuart AR, Daniel N, Joel T, et al. Production and purification of recombinant hirudin expressed in the methylotrophic yeast Pichia pastoris [J]. Protein Expression Purification, 1996, 8 : 476-482.
[7] Zhang H, Wang JD, Zhong HM, et al. Study of protein separation by reverse-phase high performance liquid chromatography [J]. Chin J Chromatogr, 1998, 16 (3) : 220-222.
[8] Zhang LX, Zhang TF, Li LY. Biochemical experimental methods and techniques [M]. 2nd ed. Beijing : Senior Education Publishing House, 2002. 137-138.
[9] Wang X, He BF, Li S, et al. Analysis of low molecular peptides by Tricine-SDS PAGE [J]. J Nanjing Univ Technol, 2003, 25 (2) : 79-81.
[10] Wei YQ, Fang HY, Yuan JR, et al. Factors affecting absorption of coptis root extract by macroporous resin [J]. Agent Tech, 2003, 12 (2) : 62-63.
[11] Loison G, Findeli A, Bernard S, et al. Expression and secretion in S. cerevisiae of biologically active leech hirudin [J]. Biotechnology, 1988, 6 :72-77.
[12] Shao L, Zhou CL, Fan W, et al. Isolation and purification of recombinant hirudin in fermentation broth [J]. J Chin Pharm Univ, 2003, 34 (3) : 268-271.

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