人胰岛素原基因在大肠杆菌中的表达和纯化

doi:10.6040/j.issn.1671-9352.0.2014.573

山东大学学报（理学版） ›› 2015, Vol. 50 ›› Issue (11): 8-15.doi: 10.6040/j.issn.1671-9352.0.2014.573

人胰岛素原基因在大肠杆菌中的表达和纯化

张晓玮¹, 彭振英¹, 陈高¹, 郑玲¹, 于金慧², 毕玉平^1,3, 边斐¹

1. 山东省农业科学院生物技术研究中心, 山东济南 250100;
2. 山东省农作物种质资源中心, 山东济南 250100;
3. 山东省农业科学院研究生教育中心, 山东济南 250100

收稿日期:2014-12-22 修回日期:2015-02-11 出版日期:2015-11-20 发布日期:2015-12-09
通讯作者: 边斐(1982-),女,助理研究员,研究方向为微生物酶学.E-mail:bxf.9@163.com;毕玉平(1961-),男,研究员,研究方向为植物生物技术与植物遗传育种.E-mail:yuping_bi@hotmail.com E-mail:bxf.9@163.com;yuping_bi@hotmail.com
作者简介:张晓玮(1990-),女,硕士研究生,研究方向为生物技术与分子生物学.E-mail:zhxw0615@126.com
基金资助:
国家国际科技合作专项资助项目(2012DFA30450);山东省自然科学基金资助项目(ZR2014CQ043);山东省农业科学院青年科研基金资助项目(2014QNZ05);山东大学微生物技术国家重点实验室开放课题资助项目(M2014-01)

Expression and purification of human proinsulin gene in Escherichia coli

ZHANG Xiao-wei¹, PENG Zhen-ying¹, CHEN Gao¹, ZHENG Ling¹, YU Jin-hui², BI Yu-ping^1,3, BIAN Fei¹

1. Biotechnology Research Center, Shandong Academy of Agricultural Sciences, Jinan 250100, Shandong, China;
2. Crop Germplasm Resources Center, Shandong Academy of Agricultural Sciences, Jinan 250100, Shandong, China;
3. Graduate Education Center, Shandong Academy of Agricultural Sciences, Jinan 250100, Shandong, China

Received:2014-12-22 Revised:2015-02-11 Online:2015-11-20 Published:2015-12-09

摘要/Abstract

摘要： 将人胰岛素原基因序列(human proinsulin, PI)经密码子优化后与TrxA蛋白融合表达,构建原核表达载体TrxA-PI转入不同大肠杆菌表达菌株BL21(DE3)、TransB(DE3)和Rosetta-gami(DE3)中。SDS-PAGE显示融合蛋白在3种表达菌株中均有表达,在Rosetta-gami(DE3)中表达量最高,是普通大肠杆菌BL21(DE3)表达量的10倍。通过优化诱导温度等发酵条件,可溶性重组融合蛋白TrxA-PI在Rosetta-gami(DE3)菌株中表达量为3.5 g/L,比未优化时提高约10倍。TrxA-PI用肠激酶酶切后,利用HisTrap FF柱分离纯化PI,经Western-blot检测重组PI具有免疫原性。

关键词: 胰岛素原, 密码子优化, 大肠杆菌表达

Abstract: The human proinsulin (PI) gene sequence was optimized using the Escherichia coli preferred codons. The reformed PI gene was amplified, the fused expression vector (TrxA-PI) was constructed and transformed into three E.coli expression strains BL21(DE3),TransB(DE3) and Rosetta-gami(DE3). SDS-PAGE analysis showed that the expression level of the fused protein was the highest in Rosetta-gami (DE3) strain, which was about 10 times higher than in BL21(DE3) strain. The production of soluble fused protein was 3.5 mg/mL after optimization of the fermentation conditions. The purified recombinant PI was obtained after enterokinase digestion and HisTrap FF column purification, and its antigen activity was measured using Western-blot analysis.

Key words: proinsulin, E.coli expression, preferred codons

中图分类号:

Q815

张晓玮, 彭振英, 陈高, 郑玲, 于金慧, 毕玉平, 边斐. 人胰岛素原基因在大肠杆菌中的表达和纯化[J]. 山东大学学报（理学版）, 2015, 50(11): 8-15.

ZHANG Xiao-wei, PENG Zhen-ying, CHEN Gao, ZHENG Ling, YU Jin-hui, BI Yu-ping, BIAN Fei. Expression and purification of human proinsulin gene in Escherichia coli[J]. JOURNAL OF SHANDONG UNIVERSITY(NATURAL SCIENCE), 2015, 50(11): 8-15.

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人胰岛素原基因在大肠杆菌中的表达和纯化

Expression and purification of human proinsulin gene in Escherichia coli

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