Eucaryotic Expression, Purification And Activity Detection Of DEK Protein

DEK is known as an abundant and ubiquitous architectural phosphoprotein in chromatin, which mainly locates in the euchromatin regions. DEK contains two functional DNA-binding domains, one is the central SAP/SAF box. domain, and the other locates on the carboxy-terminal region. DEK is a phosphoprotein with several phosphorylation sites in its carboxy-terminal region which are directly related to the function of DEK protein.Multiple reports have implicated that DEK was closely related to replication, DNA damage repair, positive and negative regulation of transcription as well as mRNA processing and differentiation. DEK is already identified as a oncogene, which is closely associated with tumorigenesis. How these activities translate into putative oncogenic DEK functions is presently unclear.We have chosen the Bac-To-Bac(?) Baculovirus Expression System to express DEK protein, and the protein is purified under native condition. In addition, we have detected its biological activity. So we have performed some basic work for further clarifying the function of DEK protein.We amplified the full-length DEK gene from pMD18-T-DEK, and then inserted it 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(?)ⅡReagent, recombinant Baculovirus (AcNPV-DEK) was developed in Sf9 cells. Then we used this viral stock to infect new Sf9 cells to generate a high-titer baculoviral stock. After that, we can use the baculoviral stock to infect Sf9 cells for DEK expression. At last, we harvested the cells at the different time points after infection (24,48,72 hours after infection). The specific 50 kD band was showed by SDS-PAGE and Western blotting, which indicated that DEK protein was expressed in Sf9 cells. The expression level of DEK protein was at the highest after infection with the recombinant virus AcNPV-DEK for 48 hours. We purified the DEK protein expressed under native conditions using Ni-NTA agarose. The analysis using SDS-PAGE and Western blotting showed that we obtained highly purified His-DEK protein.Using the purified His-DEK, we performed the Electrophoretic mobility shift assay (EMSA) 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 phosphorylated DEK protein has been reported to inhibit the binding of DEK to DNA, so we dephosphorylated the His-DEK protein usingλ-PPase. The result of EMSA showed that the DNA binding activity of dephosphorylated His-DEK protein increased than the phosphorylated protein.In summary, we expressed DEK protein by Bac-To-Bac baculovirus eukaryotic expression system and purified DEK protein with biological activity. Moreover, we tested its DNA binding activity for further clarifying the function of DEK protein.