Purification and characterization of the DeoR repressor of Bacillus subtilis

Transcription of the Bacillus subtilis dra-nupC-pdp operon Is repressed by the DeoR repressor protein, The DeoR repressor with an N-terminal His tag w as overproduced with a plasmid under control of a phage T5 promoter in Esch erichia coli and was purified to near homogeneity by one affinity chromatog raphy step. Gel filtration experimental results showed that native DeoR has a mass of 280 kDa and appears to exist as an octamer. Binding of DeoR to t he operator DNA of the dra-nupC-pdp operon was characterized by using an el ectrophoretic gel mobility shift assay, An apparent dissociation constant o f 22 nM was determined for binding of DeoR to operator DNA, and the binding curve indicated that the binding of DeoR to the operator DNG was cooperati ve. In the presence of low-molecular-weight effector deoxyribose-5-phosphat e, the dissociation constant was higher than 1,280 nM, The dissociation con stant remained unchanged in the presence of deoxyribose-1-phosphate, DNase I footprinting exhibited a protected region that extends over more than 43 bp, covering a palindrome together with a direct repeat to one half of the palindrome and the nucleotides between them.