短C肽门冬胰岛素制备工艺的研究

Research on Preparation of Insulin Aspart with Short C-Peptide

为研究门冬胰岛素制备工艺,参照毕赤酵母偏好密码子,设计合成N-端连接引导肽EEAEAEAEPK,以KEWK作为短C肽,A、B链结构完整的门冬胰岛素c DNA序列。利用Xho I-Not I酶切位点将其插入表达质粒p PICZαA,采用电转移方法将重组质粒转入毕赤酵母x-33,筛选高拷贝重组子作为工程菌株进行高密度发酵,表达量可达到0.57 g/L。发酵液经金属螯合层析和SP阳离子层析纯化后鉴定正确。建立CPB/Trypsin双酶切工艺,对不同温度及p H条件下产生的酶切产物进行分析,初步建立了酶切最佳p H值和温度条件。酶切产物经纯化后制得纯度为91.7%的门冬胰岛素,副产物为B链30位缺失的门冬胰岛素。该设计方法为含短C肽EWK门冬胰岛素制备中选用Lys-c酶切成本过高的问题提供了备选解决方案。

In order to study the preparation techniques of insulin aspart,we designed and linked EEAEAEPK to its N-terminal,referring to the preference codons of pichia pastoris,and took KEWK as C-peptide,which was a synthetise insulin aspart c DNA sequence with integrated A,B chain structure. We inserted the target sequence into expression plasmid p PICZαA by the restriction enzyme sites Xho I and Not I. The combination of P. Pastoris / x-33 was constructed by electrotransformation and the high-yield strain was selected. The combinant strain expressed 0. 57 g / L at the 64 th hours of fermentation. We obtained the purer insulin aspart precursor from broth through metal ion-affinity chromatography,SP ion-exchange chromatography and the result of authentication was correct. The system of enzyme digestion by CPB / Trypsin was established and probed the effect in different p H and temper-ature. The product after enzyme digestion was made into insulin aspart and the by-product was insulin aspart Des B30. The purity of insulin aspart reached 91. 7%. This design would be another choice to the preparation of insulin aspart,compared with which with short C-peptide EWK,as its high cost in use of Lys-c.