门冬胰岛素原的酶切及产物分离纯化方法

Method of digestion and purification of proinsulin aspart

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。

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.