Identification and purification of a DNA-binding protein interacting with the promoter of 5′-nucleotidase in Dictyostelium discoideum

The developmental management of 5′-nucleotidase (5nt) expression in Dictyostelium discoideum has provided a focal point for studies of gene regulation at the level of transcription. To identify DNA-protein interactions involved in the 5nt regulation, EMSAs were performed using short oligonucleotides, designed to span a 357 bp promoter region. A binding activity (Rf = 0.33) was identified and shown to be specific to the nucleotide sequence between −338 and −309 bp relative to 5nt ATG. Characterization of the binding activity, including the effects of salt and temperature, provided insight into the nature and stability of the protein. The protein was purified in a series of chromatographic stages, including DEAE-Sephacel, heparin-Sepharose, DNA affinity, and gel filtration. SDS-PAGE analysis identified a polypeptide with a molecular weight of 70 kDa. Mass spectrometry revealed that the purified protein was a putative formyltetrahydrofolate synthase.