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门冬胰岛素原的酶切及产物分离纯化方法
周孟能,陈文琼,陈颖,刘永东
0
(乐山市人民医院;中国科学院过程工程研究所)
摘要:
【摘要】 目的 建立门冬胰岛素原的酶切及纯化方法,获得高纯度的活性门冬胰岛素。方法 以大肠杆菌制备含有前导肽和折叠伴侣C肽的门冬胰岛素原为底物,采用胰蛋白酶和羧肽酶B对门冬胰岛素原进行酶切同时去除前导肽及C肽,酶切条件:酶与门冬胰岛素原的质量比为1:1000,室温反应30min。利用SPFF层析柱纯化酶切产物,获得高纯度的门冬胰岛素。结果 电泳结果显示酶切产物与门冬胰岛素分子量相似,离子交换纯化可以分开门冬胰岛素及酶切的多余肽段。高效凝胶过滤层析分析洗脱峰纯度大于95%,蛋白收率为30%。MALDITOF检测纯化产物的分子量与门冬胰岛素的理论分子量完全一致。结论 建立门冬胰岛素原酶切及分离纯化工艺可用于指导门冬胰岛素的规模制备。
关键词:  门冬胰岛素原  门冬胰岛素  酶切  分离纯化
DOI:
基金项目:国家自然科学基金
Method of digestion and purification of proinsulin aspart
ZHOU Mengneng,CHEN Wenqiong,CHEN Ying,LIU Yongdong
(The People's Hospital of Leshan;Institute of Process Engineering, Chinese Academy of Sciences)
Abstract:
【Abstract】 Objective To optimize the enzymatic digestion of proinsulin aspart and establish a purification protocol to obtain insulin aspart with high purity and bioactivity. Methods The enzymes are trypsin and carboxypeptidase B, and the substrate is proinsulin aspart which is expressed in E.coli with an extra peptide C to assist protein’s refolding. The reaction is operated with the mass ratio of 1: 1000 (enzyme: protein), at room temperature for 230 min. Ion exchange chromatography of SP FF was applied to purify the digested products with a gradient elution of 0100% in 15 min. Results RPHPLC and SDSPAGE analysis showed that the digestion would complete in 30 min. SP FF can separate insulin aspart from peptide C. The molecular weight of the purified insulin aspart is same as the theoretic value. The purity of insulin aspart was greater than 95% and the yield was 30%. Conclusion The enzymatic digestion and purification methods could be adopted for insulin aspart’s large production.
Key words:  Proinsulin aspart  Insulin aspart  Enzymatic digestion  Purification

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