优化雌激素受体α蛋白的可溶性表达条件  

The condition optimization of estrogen receptorαsoluble expression

作　　者：路晓庆[1] 白文启[2] 刘向荣 杨芮 张继萍[4] 郭晋锋 陈晓霞 张桓虎 Lu Xuwqing;Bai Wenqi;Liu Xiangrong;Yang Rui;Zhang Jiping;Guo Jinfeng;Chen Xiaoxia;Zhang Huanhu(Department of Breast Surgery y the Second Hospital of Shanxi Medical University,Taiyuan 030001,China;Department of General Surgery,Shanxi Cancer Hospital,Taiyuan 030001,China;The Second Clinical Medical College,Shanxi Medical University,Taiyuan 030001,China;Department of Science and Technology,the Second Hospital of Shanxi Medical University,Taiyuan 030001,China;Shanxi Cancer Hospital,Taiyuan 030001;Department of Gastroenterology,Shanxi Cancer Hospital,Taiyuan 030001,China)

机构地区：[1]山西医科大学第二医院乳腺外科,030001 [2]山西省肿瘤医院普外科,030001 [3]山西医科大学第二临床医学院,030001 [4]山西医科大学第二医院科技处,030001 [5]山西省肿瘤医院院所长办公室,030001 [6]山西省肿瘤医院消化内科,030001

出　　处：《中华实验外科杂志》2020年第7期1223-1225,共3页Chinese Journal of Experimental Surgery

基　　金：山西省科技成果转化引导专项项目(20I704D131031);山西省重点研发计划项目(201903D321162)。

摘　　要：目的构建雌激素受体α配体结合域(ERα-LBD)表达载体,优化表达条件得到可溶性ERα-LBD蛋白。方法在Addgene网站检索关键词ESR(雌激素受体,estrogen receptor),选择符合条件的质粒载体pcDNA-HA-ER WT(Addgene plasmid # 49498;http://n2t.net/addgene:49498;RRID:Addgene49498),设计引物并扩增得到目的片段ERα-LBD,分别构建蛋白表达载体pET-28a-LBD和pGEX-4T1-LBD,改变诱导温度、异丙基硫代-β-D-半乳糖苷(IPTG)浓度以及诱导时间对表达条件优化。待细菌裂解并提取蛋白后进行凝胶电泳,可在细菌上清的泳道中观察到明显的紫色蛋白表达条带,即为可溶性ERα-LBD蛋白。结果 pET-28a-LBD重组质粒未能表达重组蛋白ERα-LBD,重组质粒pGEX-4T1-LBD在Rosetta和BL21(DE3)感受态中以1 mmol/L IPTG诱导仅能得到包涵体;以0.2 mmol/L IPTG 16 ℃过夜诱导培养,则可得到可溶性ERα-LBD蛋白。结论成功构建ERα-LBD表达载体并优化诱导表达条件,获得可溶性ERα-LBD蛋白。Objective:To construct the expression vector of estrogen receptorαligand binding domain(ERα-LBD),optimize expression conditions and obtain soluble protein.Methods:The term estrogen receptor(ESR)on the Addgene was searched and pcDNA-HA-ER WT(Addgene plasmid#49498;http://n2t.net/addgene:49498;RRID:Addgene_49498)was selected.The primers were designed and the segment of ERα-LBD was obtained.pET-28a-LBD and pGEX-4T1-LBD expression vectors were constructed,and expression conditions were optimized by changing inducing temperature,time and concentration of isopropyl-β-D-thiogalactopyranoside(IPTG).After the bacteria were lysed and the protein was extracted and then subjected to gel electrophoresis,a clear purple protein expression band was observed in the lane of the bacterial supernatant,that was the soluble ERα-LBD protein.Results:Recombinant plasmid pET-28a-LBD failed to express recombinant protein ERα-LBD.Recombinant plasmid pGEX-4T1-LBD was induced by Rosetta and BL21(DE3)with 1 mM IPTG,and only inclusion bodies were obtained.With 0.2 mmol/L IPTG and cultured at 16℃overnight,soluble ERα-LBD protein was obtained.Conclusion:The ERα-LBD expression vector was constructed successfully,the inducing expression conditions were optimized and soluble protein was obtained,which built experimental operation foundation for the research of ERαsubsequently.

关 键 词：雌激素受体α配体结合域 重组表达载体 条件优化 可溶性表达 

分 类 号：Q78[生物学—分子生物学] R737.9[医药卫生—肿瘤]