Objective:To establish a reverse DNA-pull down method for verifying the binding of the target protein to the target DNA,providing a new approach for exploring the molecular mechanisms of disease development.Methods:The total cDNA of mouse bone marrow-derived macrophages was used as a template to amplify the coding sequence of the ETS domain of the transcription factor PU.1 by PCR.The ETS domain coding sequence was then ligated to the expression vector pET28a by enzymatic digestion,and then transformed into Escherichia coli BL21(DE3)for heterologous expression of the protein.The Ly96 promoter sequence was amplified with mouse genomic DNA as a template.The heterogeneously ex-pressed PU.1 ETS complex was bound to the Ni-NTA beads,after which the beads-PU.1 ETS complex was incubated with the target DNA-Ly96 promoter in vitro.The PU.1 ETS-Ly96 promoter interaction was evaluated by agarose gel elec-trophoresis after proteinase K digestion and DNA extraction to verify the feasibility of reverse DNA-pull down.Results:The Ni-NTA beads-PU.1 ETS complex was obtained through heterologous expression and affinity purification.The Ly96 promoter sequence was amplified by PCR,and the interaction between PU.1 ETS and the Ly96 promoter was verified by reverse DNA-pull down.Conclusion:Taking the interaction of PU.1-ETS-Ly96 promoter as an example,the feasibility of reverse DNA-pull down technology is confirmed,which provides an alternative scheme with simple operation,low experi-mental conditions and low cost for exploring the molecular mechanism of binding of known protein to target DNA and dis-ease development.
protein-DNA bindingreverse DNA-pull down assayheterologous expressionPU.1 transcription factor
万方数据
维普
