摘要
为了制备GST-Exendin-4融合蛋白,以本实验室构建好的pET22b-Exendin-4为模板,通过PCR扩增出Exendin-4基因,酶切克隆入pGEX-4T-1,构建重组表达质粒pGEX-4T-1-Exendin-4。经DNA测序证实插入序列与设计完全一致后,将重组质粒转化大肠杆菌BL21(DE3),在不同浓度的IPTG和不同温度诱导下,经SDS-PAGE分析鉴定,表达出Mr约为30 kD的目的蛋白,灰度扫描显示目的蛋白占菌体总蛋白的29.9%,超声裂解后的上清通过Sepharose 4B亲和层析纯化、透析、除盐及冷冻干燥得到高纯度的目的蛋白。本研究成功制备了高纯度的GST-Exendin-4融合蛋白,为下一步进行目的蛋白大规模的生产打下了良好的基础。
For the sake of a high expression of Exendin-4 in E. coli, Exendin-4 was synthesized by PCR with the template of pET22b-Exendin-4 ,which was previously constructed in the key lab of Plant Genetics ＆ Molecular Breeding in Zhoukou Normal Uni- versity. The PCR product was cloned into pGEX-4T-1 vector and the positive clones were confirmed by sequencing. The recombinant vector pGEX-4T-1-Exendin-4 and empty vector pGEX-4T-lwere transformed into E. coli strain BI21 （ DE3 ） respectively. Then the 1 mmol/L IPTG and 0.5 mmol/L IPTG induction under 37℃ and 25℃ was tested using SDS-PAGE analysis separately and the fusion protein GST-Exendin-4 of Mr 30 kD was inquired. It was estimated by gray scale scanning method that 29.9% of the total bacterial pro- tein is the fusion protein GST-Exendin-4. The high-purity recombinant GST-Exendin-4 fusion protein was obtained through glutathione affinity purification using Sepharose 4B affinity column,desalination and cryodesiccation. In our research,the high-purity soluble fusion protein GST-Exendin-4 can be expressed in E. coli benefiting future large-scale production of Exendin-4.
出处
《生物技术通报》
CAS
CSCD
北大核心
2011年第6期155-159,共5页
Biotechnology Bulletin
基金
国家自然科学基金资助项目(30901877)
河南省科技厅社会发展项目(092102310209)
河南省教育厅自然科学研究计划项目(2009B180021)
