人转录因子USF基因在大肠杆菌中的表达、纯化及在FAK启动子中的结合研究

Expression,purification of human transcription factor USF and its DNA binding analysis in FAK promoter

目的:在大肠杆菌中表达人源转录因子USF(upstream stimulatory factor)并进行分离纯化,进一步分析其在FAK(focaladhesion kinase)启动子中的DNA结合能力。方法:将人源USF cDNA克隆到表达质粒pET28a载体,然后将重组表达质粒转化大肠杆菌BL21(DE3),经IPTG诱导表达带His6-Tag的融合蛋白His-USF,通过镍柱亲和层析纯化,SDS-PAGE分析鉴定。EM-SA分析纯化后的蛋白与FAK启动子的DNA结合情况。结果:His-USF在大肠杆菌中获得高效表达,镍柱亲和层析纯化得到了高纯度的蛋白,并能够结合到FAK的启动子上。结论:获得高效表达的高纯度His-USF融合蛋白,为USF对其靶基因的结合与转录调控的进一步研究奠定了基础。

Objectlve To express the gene encoding human USF（ upstream stimulatory factor） in E. coli, and analyze its DNA binding in FAK promoter. Method The human cDNA was cloned into vector pET28a, then transformed into E. coli BL21 （ DE3 ） for expression. The fusion protein His-USF was expressed when induced by adding of IPTG and purified with Ni-IDA. SDS-PAGE was used to detect the expressed protein. Then we analyzed the DNA binding ability of the purified protein in FAK（focal adhesion kinase） promoter using EMSA assay. Result A high level expression of His-USF fusion protein was obtained and purified. The purified protein could bind the FAK promoter. Conclusion His-USF fusion protein is expressed with high efficiency in prokaryotic expression system, providing a foundation of the further study of USF transcriptional regulation on its target gene.