Expression,purification And Activity Assay Of DNA Binding Proteins

Background and Objective:The research on DNA binding proteins is becoming a trend because of their key role in replication,recombination and expression of DNA.DNA-binding proteins include transcription factors,DNA/RNA polymerases,nucleases,histones and histone-like proteins in all living cells.DNA binding proteins can be divided into three classes according to its binding properties with DNA: those specifically bind single-stranded DNA,e.g.SSB protein,those non-specifically bind DNA,e.g.HU protein,H-NS protein,IHF protein and Fis protein,and those bind to specific DNA sequences,e.g.DNA polymerases and endonucleases.The study of DNA binding proteins from Escherichia coli is most comprehensive at present.Escherichia coli SSB(ecSSB)is now known to bind to ssDNA and interact with at least 14 other proteins that involved in DNA metabolism in vivo?EcSSB can improve the performance of PCR,capillary electrophoresis and pyrosequencing,it can also decrease nonspecific amplication of RCA.HU and Fis from Escherichia coli are involved in chromosome packaging as global regulators.To gain a better understanding of the features of these proteins,a fast and easy purification method is required.Method:Here we report a column-free purification method of DNA binding proteins which is resin-independent and tag-less by using PEI and ammonium sulfate only.1)Construct expression vectors:pET22b-ecSSB-His ? pET22b-HU?-His and pET22b-Fis.2)PEI and ammonium sulfate are applied for column-free purification of ecSSB-His,HU?-His and Fis based on characteristics of reagent-protein interaction.Then the purified proteins and their binding activities were identified respectively.3)EcSSB-His,HU?-His were purified through nickel column and then compare with the column-free method.Results:1)We successfully purified ecSSB-His,HU?-His and Fis proteins through column-free purification method using PEI and ammonium sulfate.2)We also obtain ecSSB-His and HU?-His proteins through nickel column.Both of the purified proteins are of high purity.But the column-free purification method has advantages of tag-less-protein,easy-to-oeprate and low-cost.