DEK蛋白的真核表达纯化及其活性检测

中国生物工程杂志

2011, Vol. 31

Issue (12): 1-9

研究报告

DEK蛋白的真核表达纯化及其活性检测

郭海红, 杨辛, 郭芳, 胡红刚

北京交通大学理学院生命科学与生物工程研究院北京 100044

Eucaryotic Expression and Purification of DEK Protein and Its Activity Detection

GUO Hai-hong, YANG Xin, GUO Fang, HU Hong-gang

College of Life Sciences and Bioengineering, School of Science, Beijing Jiaotong University, Beijing 100044, China

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摘要：

利用Bac-to-Bac杆状病毒表达系统表达DEK蛋白并进行纯化。首先以pFastBacI质粒构建重组质粒pFastBacI-DEK,转化DH10Bac大肠杆菌后获得重组穿梭载体Bacmid-DEK,通过脂质体介导转染Sf9细胞产生具有强感染力的重组杆状病毒AcNPV-DEK。用此重组杆状病毒AcNPV-DEK感染Sf9细胞表达His-DEK融合蛋白。在非变性条件下,利用Ni-NTA agarose对表达的His-DEK融合蛋白进行纯化,经SDS-PAGE和Western blotting分析,在50 kDa处出现特异性蛋白条带并证实其为His-DEK融合蛋白。凝胶迁移阻滞实验表明,融合蛋白His-DEK与DNA 的结合具有结构特异性,其与超螺旋型DNA结合活性强于与线性化DNA的结合活性。真核表达并纯化的融合蛋白His-DEK与DNA的结合活性要明显强于原核表达的融合蛋白His-CDB。DEK 蛋白的磷酸化修饰会阻碍其与DNA的结合,而Sf9细胞中表达的融合蛋白His-DEK存在磷酸化修饰,将His-DEK去磷酸化后,其与DNA的结合活性有所提高。

关键词： DEK蛋白; 杆状病毒表达系统; 凝胶迁移阻滞

Abstract:

It was chosen the Bac-To-Bac^® Baculovirus Expression System to express DEK protein, and the protein was purified under native condition. First, the full-length DEK gene was amplified from pMD18-T-DEK, and then inserted into plasmid pFastBacI of the Bac-To-Bac^® Baculovirus expression system to form plasmid pFastBacI-DEK. Recombinant shuttle vector named Bacmid-DEK was obtained in E.coli DH10Bac through transformation. After transfection with Cellfectin^® II Reagent, recombinant Baculovirus (AcNPV-DEK) was developed in Sf9 cells. Then this viral stock was used to infect new Sf9 cells to generate a high-titer baculoviral stock. After that, the baculoviral stock can be used to infect Sf9 cells for DEK expression. The DEK protein was purified under native conditions using Ni-NTA agarose. The specific 50 kDa band was showed by SDS-PAGE and Western blotting, which indicated that DEK protein was expressed in Sf9 cells and the highly purified His-DEK proteins were obtained. Using the purified His-DEK, the Electrophoretic mobility shift assay (EMSA)was performed to test the binding activity of His-DEK to DNA molecules. The results showed that His-DEK and His-CDB (expressed by E.coli BL21 strain) could bind DNA molecules, especially, preferring the supercoiled form in vitro; meanwhile, the DNA binding activity of His-DEK was higher than His-CDB. It is reported that phosphorylation of DEK protein could inhibit the binding of DEK to DNA. The expressed His-DEK protein in Sf9 cells was phosphorylated, so the His-DEK protein was dephosphorylated using λ-PPase. The result of EMSA showed that the DNA binding activity of dephosphorylated His-DEK protein increased than the phosphorylated protein.

Key words: DEK protein Baculovirus expression system Electrophoretic mobility shift assay

收稿日期: 2011-08-22 出版日期: 2011-12-25

ZTFLH:

Q819

基金资助:

教育部留学回国人员基金资助项目(S09C300050)

通讯作者: 胡红刚,电子信箱:hghu@bjtu.edu.cn E-mail: hghu@bjtu.edu.cn

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引用本文:

郭海红, 杨辛, 郭芳, 胡红刚. DEK蛋白的真核表达纯化及其活性检测[J]. 中国生物工程杂志, 2011, 31(12): 1-9.

GUO Hai-hong, YANG Xin, GUO Fang, HU Hong-gang. Eucaryotic Expression and Purification of DEK Protein and Its Activity Detection. China Biotechnology, 2011, 31(12): 1-9.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I12/1

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