人转录因子sp1在大肠杆菌中的表达与体外纯化
Expression in E.coli and purification in vitro of human transcription factor sp1
王琪炜 1陈宝俊 2强倩 1李淑锋3
作者信息
- 1. 东南大学生命科学研究院,江苏南京210018
- 2. 南京大学医学院附属鼓楼医院心胸外科,江苏南京210008
- 3. 东南大学医学院生物化学与分子生物学系,江苏南京210009
- 折叠
摘要
目的:在大肠杆菌中表达人源转录因子sp1蛋白,并进行体外纯化.方法:首先将人源sp1真核表达质粒pCMV-sp1中的sp1 cDNA用限制性内切酶切开,连入原核表达质粒pET28b,构建原核表达重组质粒pET28b-sp1-699c;然后将重组质粒转化入大肠杆菌BL21(DE3)菌株,经IPTG诱导表达带His-Tag的融合蛋白His-sp1 (88-786aa),通过包涵体的变复性和Ni-IDA亲和层析纯化后,SDS-PAGE鉴定纯化后的蛋白.结果:融合蛋白His-sp1在大肠杆菌内得到高效表达,纯化后可得到较高纯度的蛋白.结论:本研究获得了较高纯度的融合蛋白His-sp1,为进一步研究sp1蛋白对靶基因的转录调控奠定了基础.
Abstract
Objective:To express human transcription factor sp1 in E.coli and purify this protein.Methods:(1) Prokaryotic expression recombinant plasmid pET28b-sp1-699c was constructed,while cutting the cDNA of sp1 from human sp1 eukaryotic expression plasmid pCMV-sp1 with restriction enzymes and inserting into prokaryotic expression plasmid pET28b by T4 DNA ligase.(2) This recombinant plasmid was transform into BL21 (DE3) E.coli strain,expression of His-Tag fusion protein His-sp1 with IPTG induction.(3) The products by denaturation and renaturation of inclusion body,and affinity chromatography were purified,and then the purified protein was detected by SDS-PAGE.Results:Fusion protein His-sp1 was expressed efficiently in E.coli,and protein with high purity was obtained.Conclusion:In this study,we obtained fusion protein His-sp1 with high purity and quantity,laid a foundation for further research of the transcription regulation sp1 target gene.
关键词
sp1蛋白/转录因子/大肠杆菌/蛋白表达与纯化Key words
sp1 protein/transcription factor/E.coli/expression and purification of protein引用本文复制引用
基金项目
国家自然科学基金面上项目(31070706)
出版年
2014
NETL
NSTL
维普
万方数据