Refolding, purification, and preliminary structural characterization of the DNA-binding domain of the quorum sensing receptor RhlR from Pseudomonas aeruginosa

• RhlR DBD was refolded in the presence of the charged amino acids l-arginine and l-glutamate.
• 2 mg/L culture of highly purified RhlR DBD were obtained.
• RhlR DBD is folded and monomeric.
• RhlR DBD is functional as it is capable of recognizing the rhlAB promoter.
• This refolding and purification protocol will enable further biochemical and structural studies on RhlR DBD.

RhlR is a 241-residue quorum sensing receptor that controls the expression of a myriad of virulence genes in Pseudomonas aeruginosa. Here, the DNA sequence encoding the carboxi-terminal DNA-binding domain of RhlR was cloned into the pET-RP1B plasmid and expressed as an N-terminal fusion protein to the expression/purification Thio6His6 tag. The fusion construct expressed insolubly in Escherichia coli BL21 (DE3) cells. The recombinant protein was extracted from the bacterial inclusion bodies and refolded in the presence of the charged amino acids l-arginine and l-glutamate. The refolded protein was purified by a combination of Ni+2-affinity and size exclusion chromatography, allowing the production of 2 mg of highly purified protein (>95% purity) per 5 mg of wet cells derived from 1 L culture. 1H 1D NMR analysis revealed that the recombinant protein is folded. Moreover, a fluorescence anisotropy DNA-binding assay showed that the refolded protein is functional, as it recognizes the rhlAB promoter. This is the first time that a domain of the quorum sensing regulator RhlR was produced in sufficient amounts for structural studies, enabling the investigation of the molecular basis for RhlR specific interaction with DNA promoters.