Purification and characterization of a regulatory protein XyIR in the D-xylose operon from Escherichia coli

The D-xylose operon in Escherichia coli is known to be regulated by a transcriptional activator protein, XylR, which is responsible for the expression of both xylAB and xylFGH gene clusters. The XylR was purified to homogeneity by using the maltose binding protein fusion expression and purification systems involving two chromatography steps. The purified XylR protein was composed of two subunits of 45 kDa, which was determined by both sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration. The purified XylR was specifically bounded to the xylA promoter, regardless of adding xylose to the reaction mixture, but binding of XylR to the xylA promoter was enhanced by adding xylose. The enhanced binding ability of XylR in the presence of xylose was not diminished by adding glucose. The presumed XylR binding site is located between 120 bp to 100 bp upstream from the xylA initiation codon.