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

N-Terminal Heterogeneity and Purification of Insulin Precursor Expressed in Pichia pasoris

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.