人转录因子sp1在大肠杆菌中的表达与体外纯化-东南大学学报医学版

人转录因子sp1在大肠杆菌中的表达与体外纯化

单位：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

分类号：Q876

出版年·卷·期（页码）：2014·33·第一期（1-4）

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。

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.

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