利用GST融合蛋白表达系统表达与纯化人N-Ras蛋白  

Expression and Purification of Human N-Ras Protein with Fused GST Protein Expression System

作　　者：张云静[1,2] 冯滢滢[2] 徐小洁[1] 梁迎春[1] 张立 王涛[2] 李玲[1] 郭靖[1] 纪贝贝 张蓉[2] 叶棋浓[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100850 [2]第二炮兵总医院,北京100088 [3]总参警卫局卫生保健处,北京100017

出　　处：《生物技术通讯》2015年第4期497-500,共4页Letters in Biotechnology

基　　金：国家自然科学基金(81472589;31100604);北京市自然科学基金(7132155);北京市科技新星计划(Z141102001814055);军事医学科学院创新基金转化医学项目(ZHYX003)

摘　　要：目的:克隆人N-ras蛋白全长编码区基因,获得其原核表达产物,并对融合蛋白进行纯化。方法:采用PCR技术从人乳腺文库中扩增出人N-ras蛋白全长编码区基因,将其克隆到p GEX-KG载体中,在大肠杆菌Rossate中表达后,利用GST-Sepharose 4B亲和珠对原核表达产物进行纯化,SDS-PAGE鉴定表达与纯化产物。结果:从人乳腺文库中扩增获得约600 bp的DNA片段,并克隆至p GEX-KG载体上,经测序与目的序列完全一致;在大肠杆菌Rossate中诱导表达出相对分子质量约47×103的目的蛋白;纯化后,经鉴定获得了纯度较高的重组蛋白GST-N-Ras。结论:获得了重组蛋白GST-N-ras,为后续深入研究Ras基因与其他癌基因、抑癌基因的相互作用奠定了基础。Objective：To construct the prokaryotic expression vector of human N-Ras gene,obtain the purifiedGST-N-Ras protein.Methods：Human N-Ras gene coding region was amplified from human mammary DNA li-brary by the technique of PCR,and was inserted into prokaryotic expression vector p GEX-KG.The recombinantplasmid p GEX-KG-N-Ras was transformed into E.coli Rossate.The expressed product was purified by GST-Sepha-rose 4B beads and identified by SDS-PAGE analysis.Results：The DNA fragment of N-Ras about 600 bp wasamplified by PCR,successfully cloned into p GEX-KG and identified by sequencing.The recombinant protein GST-N-Ras with Mr 47 k D was successfully induced,purified well.Conclusion：The recombinant protein of GST-N-Ras is obtained successfully,which lay the foundation for further research between Ras gene and other gene,in-cluding oncogene and tumor suppressor gene.

关 键 词：人N-ras蛋白 小G蛋白 原核表达 纯化 

分 类 号：Q78[生物学—分子生物学]