文献类型：期刊文献

中文题名：毕赤酵母表达人胰岛素前体的纯化和N末端不均一性分析

英文题名：N-Terminal Heterogeneity and Purification of Insulin Precursor Expressed in Pichia pasoris

作者：刘海峰[1,2,3];周祥山[1];庞甲佩[2];刘立岗[2];胡永水[3];张元兴[1]

机构：[1]华东理工大学生物反应器工程国家重点实验室,上海200237;[2]山东阿华生物药业有限公司,山东聊城252201;[3]山东东阿阿胶股份有限公司,山东聊城252201

年份：2013

卷号：39

期号：5

起止页码：565

中文期刊名：华东理工大学学报（自然科学版）

外文期刊名：Journal of East China University of Science and Technology

收录：CSTPCD;;Scopus;北大核心:【北大核心2011】；CSCD:【CSCD2013_2014】；

基金：国家科技重大专项(2011ZX09202-301-05)

语种：中文

中文关键词：毕赤酵母;胰岛素前体;反相层析;MALDI—TOF—MS;N末端测序

外文关键词：Pichiapastoris; insulin precursor（IP）; revered phase chromatography（RPC）; MALDI- TOF-MS; N terminal sequencing

摘要：利用毕赤酵母生产人胰岛素前体时,在胰岛素前体的N末端引入一段由lO个氨基酸组成的间隔肽(EEAEAEAEPK)虽然可以提高目的产物的表达量,但间隔肽中的多个酶切位点有可能造成目的产物N末端的不均一性。对毕赤酵母分泌表达的胰岛素前体蛋白进行分离纯化,得到的样品经过MALDI-TOF-MS和N末端测序,证实了胰岛素前体N末端不均一性的现象,并对产生不均一性的原因进行了分析。实验同时证实,利用毕赤酵母得到的胰岛素前体,经胰蛋白酶酶切后,生成了B链缺少第30位苏氨酸但N末端均一的胰岛素产物desB30,且C末端未发生降解,这为人胰岛素和地特胰岛素的制备提供了新思路。
Aiming to improve the fermention yield of insulin precursor （IP） in Pichia pastoris, a spacer peptide of 10 amino acids （EEAEAEAEPK） was introduced into the N-terminal of the IP. This sequence might cause the possibility of the N-terminal heterogeneity of the products. The IP produced in Pichia pastoris was purified by ion-exchange chromatography and reversed phase chromatography. The purified samples were analyzed with MALDI-TOF-MS and N-terminal sequencing. Results proved the existence of N-terminal heterogeneity and helped to find the possible causes for this phenomenon. The results also indicated that the IP produced by P. pastoris could generate a mutated insulin with deletion of threonineB30（desB30） in chain B which posessed the characteristics of N-terminal homogeneity and no degradation in C-terminal after digested by trypsin. This research provided a new method for human insulin and detemir insulin production.