病原诱导的小麦ERF转录因子TaERF1b的原核表达及纯化  

Expression and Purification of Pathogen-induced Wheat ERF Transcription Factor TaERF1b in Escherichia coli

作　　者：董娜[1] 张增艳[1] 辛志勇[1] 

机构地区：[1]中国农业科学院作物科学研究所/农作物基因资源与基因改良国家重大科学工程/农业部作物遗传育种重点开放实验室,北京100081

出　　处：《植物遗传资源学报》2008年第3期283-287,共5页Journal of Plant Genetic Resources

基　　金：国家自然科学基金(30671292)

摘　　要：为了得到纯化的TaERF1b活性蛋白,将TaERF1 b基因含有AP2/ERF结构域的片段插入原核表达载体pGEX-4T-1的多克隆位点中,构建GST-TaERF1b融合蛋白表达载体,并转化到大肠杆菌BL21(DE3)中。0.1mmol/L IPTG即能诱导融合蛋白表达,37℃诱导4h或30℃诱导8h,融合蛋白均以包涵体的形式表达,16℃诱导12h,融合蛋白不表达。包涵体经溶解及稀释复性后,过GST亲和层析柱,获得纯化的融合蛋白,考马斯亮蓝法测得纯化蛋白的浓度约为0.5μg/μl,凝胶阻滞实验表明包涵体复性成功,所得蛋白具有生物活性。To get purified and active TaERF1 b protein, the fragment encoding AP2/ERF domain of TaERF1b was fused into the BamHI-SalI sites of the pGEX-4T-1 vector in a frame with GST, and transformed into Escherichia coli BL21 （DE3） cells. The fusion protein could be induced to express by 0.1 mmol/L IPTG, in the form of inclusion body at 37℃ for 4h or 30℃ for 8h, but could not be induced at 16℃ for 12h. Following denaturation and renaturation of inclusion body, the renatured protein was purified using MicroSpinTM G-50 columns. The concentration of purified protein was about 0.5ug/ul. Finally, the purified protein was confirmed to be of bioactivity by electrophoretic mobility shift assay.

关 键 词：ERF(ethylene-responsive factor) TaERFlb 原核蛋白表达 包涵体 

分 类 号：S435.121[农业科学—农业昆虫与害虫防治]