一种新的B链C端去四肽胰岛素的研制方法

A New Method for Preparation of Monomeric Destetrapeptide Human Insulin

报道了将单体胰岛素前体(MIP)经胰蛋白酶和羧肽酶B两步连续酶切获得B链C端去四肽胰岛素(DTI)的方法。MIP由甲醇酵母表达,最高发酵表达量达到150mg/L。发酵液中MIP通过疏水层析,分子筛初步纯化后直接进行酶切,在胰蛋白酶酶切3h后加入抑制剂paminobenzamidine处理15min,然后直接加入羧肽酶B酶切6h,再通过反相柱纯化即可得到纯品DTI,从分子筛到最后DTI,总纯化得率达到77%。按中国药典小白鼠惊厥法测定得DTI的生物活力为22IU/mg,是胰岛素的80%,在Superdex G-75分子筛上测定DTI的解离聚合曲线,证明其是单体。

Monomeric destetrapeptide human insulin （DTI, human insulin with B27-30 removed） was obtained from a monomeric insulin precursor （MIP） expressed in Picha pastoris through two step subsequent hydrolysis with trypsin and carboxypeptidase B. The crude MIP which purified by hydrophobic and size-exclusion chromatography was converted to be DTI by hydrolysis with trypsin for 3 h and with carboxypeptidase B for 6 h. Before hydrolysis with carboxypeptidase B, the activity of trypsin was inhibited by adding inhibitor p-aminobenzamidine for 15 min. The crude DTI was then purified by reverse processing chromatography. The yield of MIP was 150 mg per liter of culture, and the overall yield of purified DTI from crude MIP was 77%. The in vivo biological activity of DTI as determined by the mouse convulsion assay was 22 IU/mg. Compared with native insulin, DTI molecules do not aggregate in solution but exist in the monomeric form.