DEK蛋白的真核表达纯化及其活性检测

Eucaryotic Expression and Purification of DEK Protein and Its Activity Detection

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。

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.