重组猪促肾上腺皮质激素在大肠埃希菌中的高效表达与分离纯化  

High-level Expression and Purification of Recombinant Pig Adrenocorticotropic Hormone in Escherichia coli

作　　者：黄宗庆 化浩举 陆伟根 吴勇 HUANG Zongqing;HUA Haoju;LU Weigen;WU Yong(Shanghai Duomirui Bio-Technology Co.,Ltd.,Shanghai 201203;China State Institute of Pharmaceutical Industry,Shanghai 201203)

机构地区：[1]上海多米瑞生物技术有限公司,上海201203 [2]中国医药工业研究总院,上海201203

出　　处：《中国医药工业杂志》2020年第3期334-339,共6页Chinese Journal of Pharmaceuticals

基　　金：国家“重大新药创制”科技重大专项(2018ZX09721003-008-013)。

摘　　要：本研究建立了一种以大肠埃希菌作为宿主,表达重组猪促肾上腺皮质激素(pig adrenocorticotropic hormone,pACTH)的新方法。pACTH在N末端与经典猪瘟病毒N末端自切蛋白酶Npro突变体EDDIE融合,然后在大肠埃希菌胞浆中以包涵体形式表达,包涵体约占菌体湿重的36.2%。经过复性后的包涵体可以促使EDDIE自酶切,从而获得pACTH。通过优化复性条件,EDDIE-pACTH蛋白的自切率从25%提高至60%。复性液通过酸沉淀,能够去除杂蛋白,经反相色谱纯化后获得纯度为99.56%的pACTH。This study described a novel approach for the large scale production of recombinant pig adrenocorticotropic hormone(pACTH)in Escherichia coli.The pACTH was fused at the N-terminus to the N-terminal autoprotease Npro mutant EDDIE from classical swine fever virus,and was expressed in the E.coli cytoplasm in inclusion bodies at levels about 36.2%of the wet cell weight.The renatured inclusion bodies were able to induce EDDIE autoproteolysis to obtain pACTH.By optimizing the refolding conditions,the self-cleavage rate of EDDIE-pACTH protein was increased from 25%to 60%.The refolding solution was subjected to one-step acid precipitation to remove the heteroprotein,and purified by reverse phase chromatography to obtain pACTH having a purity of 99.56%.

关 键 词：促肾上腺皮质激素 自切蛋白酶 自我切割 重组表达 

分 类 号：TQ464.7[化学工程—制药化工] Q816[生物学—生物工程]