title: Refolding, purification, and preliminary structural characterization of the DNA-binding domain of the quorum sensing receptor RhlR from Pseudomonas aeruginosa.
abstract: 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. (1)H 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.
authors.affiliation: Department of Biochemistry, Institute of Chemistry, Federal University of Rio de Janeiro, Rio de Janeiro 21941-909, Brazil.
authors.forename: Carolina
authors.initials: C
authors.lastname: Lixa
authors.affiliation: School of Pharmacy, Federal University of Rio de Janeiro, Rio de Janeiro 21941-590, Brazil.
authors.forename: Adriana F
authors.initials: AF
authors.lastname: Marques
authors.affiliation: Department of Virology, Institute of Microbiology Paulo de Goés, Federal University of Rio de Janeiro, Rio de Janeiro 21941-902, Brazil.
authors.forename: Juliana R
authors.initials: JR
authors.lastname: Cortines
authors.affiliation: Department of Biochemistry, Institute of Chemistry, Federal University of Rio de Janeiro, Rio de Janeiro 21941-909, Brazil.
authors.forename: Bianca C
authors.initials: BC
authors.lastname: Neves
authors.affiliation: Department of Biochemistry, Institute of Chemistry, Federal University of Rio de Janeiro, Rio de Janeiro 21941-909, Brazil.
authors.forename: Danielle M P
authors.initials: DM
authors.lastname: Oliveira
authors.affiliation: Department of Biochemistry, Institute of Chemistry, Federal University of Rio de Janeiro, Rio de Janeiro 21941-909, Brazil.
authors.forename: Cristiane D
authors.initials: CD
authors.lastname: Anobom
authors.affiliation: School of Pharmacy, Federal University of Rio de Janeiro, Rio de Janeiro 21941-590, Brazil.
authors.forename: Luís Maurício T R
authors.initials: LM
authors.lastname: Lima
authors.affiliation: Department of Biochemistry, Institute of Chemistry, Federal University of Rio de Janeiro, Rio de Janeiro 21941-909, Brazil. Electronic address: pinheiro@iq.ufrj.br.
authors.forename: Anderson S
authors.initials: AS
authors.lastname: Pinheiro
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D011401:Promoter Regions, Genetic; D017510:Protein Folding; D011550:Pseudomonas aeruginosa / Q000235:genetics; D053038:Quorum Sensing / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C086515:RhlR protein, Pseudomonas aeruginosa
keywords: Purification; Quorum sensing; Receptor; Refolding; RhlR