Expression And Purification Of DEK Protein And Preliminary Study On Its Function

DEK is known as an abundant and ubiquitous architectural phosphoprotein in chromatin.Previous studies show that DEK is composed of 375 amino acids,which mainly locates on the euchromatin regions.Two functional DNA-binding domains were found in its amino acid sequences,one is the SPA/SAF box domain,another is the Carboxyl-terminal DNA binding region(CDB).There are multiple phosphorylated sites in its CDB domain.The phosphorylation of these sites are directly related to the function of DEK protein.The overexpression of this protein was found in cells of some autoimmune diseases and some human tumor cell lines.It is also found the low expression in certain malignancy tumor cells.The increasing data show that there are some kinds of links between the apoptosis and DEK protein.Presently,the exact functions of DEK protein are not known clearly.From the analysis of DEK studies,the CDB of DEK may play important roles for the functions of DEK.In this study,we selected the CDB domain as the major functional region which may provide some clues for understanding the biological functions of DEK protein.Using the cDNA of Hela cells,we first generated two constructs,pMD18-T-DEK and pET-30a(+)-CDB.The His-tagged CDB and DEK were purified from the E.coli BL21 strain.Using the purified His-CDB,we performed the Electrophoretic mobility shift assay(EMSA) to test the binding activity between CDB and DNA molecules.The results showed that CDB can bind DNA molecules,especially,preferring the supercoiled form in vitro,which is similar to the DNA binding pattern of the full length DEK protein.To test the effect of the different regions of DEK on cell proliferation and apoptosis, we created the constructs with full length and different regions of DEK gene for transfection of Hela cell lines.After transfecting Hela cells with these constructs,we measured and calculated the relationships between the protein expression and the cell proliferation activities or apoptosis following the time points.The results showed that full length DEK,CDB with NLS,and CDB might affect the cell proliferation or apoptosis,suggesting that the further experiments need to confirm the results above.To further test the expression of DEK protein in different cell lines,we performed the western blotting to examine the level of this protein.The expression of DEK in these six cell lines exhibited different levels,A549 is very low compared with those of other cell lines,indicating that the modification of this protein and transcription regulation of this protein need further exploration.