GST-Smad4融合蛋白的表达与纯化  

Prokaryotic Expression and Purification of GST-Smad4 Protein

作　　者：梅竹[1] 杨予涛[1] 徐志卿[1] 

机构地区：[1]首都医科大学基础医学院,北京100069

出　　处：《生物技术通报》2013年第1期134-138,共5页Biotechnology Bulletin

基　　金：国家"973"计划项目(2010CB912003);高等学校博士学科点专项科研基金项目(20101107120015);北京市属高等学校人才强教深化计划中青年骨干人才资助项目(PHR20110895);北京市优秀人才培养资助项目(2010D005018000010)

摘　　要：旨在原核表达Smad4基因,纯化获得GST-Smad4融合蛋白。以人表皮HaCaT细胞的cDNA为模板,利用PCR扩增含有BamH I和SalI酶切位点的Smad4基因;然后将其克隆到pGEX-4T-1原核表达载体中,将正确的重组载体转入大肠杆菌BL21(DE3);用IPTG诱导表达,再利用MagneGST particles亲和纯化GST-Smad4融合蛋白;最后通过Western blot鉴定此融合蛋白。结果显示,成功构建pGEX-4T-1-Smad4原核表达载体;30℃条件下,0.2 mmol/L的IPTG能诱导出大量的可溶性GST-Smad4蛋白;经MagneGST particles纯化的GST-Smad4蛋白可被Smad4的抗体特异识别。纯化的GST-Smad4蛋白可用于后续的生物学研究。It was to express human Smad4 gene in prokaryotic cells and purify the GST-Smad4 fusion protein. The full-length Smad4 gene was amplified by PCR and cloned into prokaryotic expression vector pGEX-4T-1. The recombinant plasmid pGEX-4T-1-Smad4 was transformed into E.coli BL21 （ DE3 ） and exogenous protein was induced by IPTG. After purification using MagneGST particles, the GST-Smad4 fusion protein was further identified by Western blot. Results showed that the recombinant plasmid pGEX-4T-1-Smad4 was constructed successfully. When BL21 （ DE3 ） cells transformed with pGEX-4T-1-Smad4 were cultured at 30℃ and induced with 0.2 mmoFL IPTG, GST-Smad4 protein was obtained in a large quantity in supernatant. The purified GST-Smad4 was further identified specifically by Smad4 antibody. Therefore, it proved that GST-Smad4 fusion protein was successfully expressed and purified, and could be used for further study of the function of Smad4.

关 键 词：SMAD4 原核表达 蛋白纯化 

分 类 号：Q78[生物学—分子生物学]