Purification and characterization of Pseudomonas aeruginosa LasR expressed in acyl-homoserine lactone free Escherichia coli cultures

Protein Expr Purif. 2017 Feb;130:107-114. doi: 10.1016/j.pep.2016.10.007. Epub 2016 Oct 15.


Quorum sensing systems are essential for bacterial communication. We report here the purification and characterization of the Pseudomonas aeruginosa LasR quorum sensing regulator purified from lysates of E. coli cultures grown in the absence of added acyl-homoserine lactones (AHL). We show by isothermal titration calorimetry that LasR recognizes different AHLs with an affinity of approximately 1 μM. The affinity of LasR for its cognate 3-Oxo-C12-AHL was similar to that of other AHLs, indicating that this regulator has not evolved to preferentially recognize its cognate AHL. The α-helical content as determined by CD spectroscopy was found to be in agreement with the corresponding value derived from the homology model. Analytical ultracentrifugation studies show that LasR is a mixture of monomers and dimers and that AHL binding does not alter its oligomeric state. Thermal unfolding studies indicate that LasR has a significant thermal stability and that AHL binding does not significantly alter the unfolding temperature. Two LasR-DNA complexes were observed in electrophoretic mobility shift assays using the hcnABC promoter that has two lux boxes. Taken together, data indicate that the presence of AHLs is not a requisite for correct LasR protein folding. The protein is able to bind AHL ligands in a reversible manner, revising initial concepts of this regulator. The availability of AHL-free protein will permit further studies to determine more precisely its mode of action.

Keywords: Acyl-homoserine lactone; Quorum sensing; Transcriptional regulator.

MeSH terms

  • Acyl-Butyrolactones / chemistry*
  • Bacterial Proteins* / biosynthesis
  • Bacterial Proteins* / chemistry
  • Bacterial Proteins* / genetics
  • Bacterial Proteins* / isolation & purification
  • Escherichia coli / genetics
  • Escherichia coli / growth & development*
  • Pseudomonas aeruginosa / genetics*
  • Pseudomonas aeruginosa / metabolism
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Trans-Activators* / biosynthesis
  • Trans-Activators* / chemistry
  • Trans-Activators* / genetics
  • Trans-Activators* / isolation & purification


  • Acyl-Butyrolactones
  • Bacterial Proteins
  • LasR protein, Pseudomonas aeruginosa
  • Recombinant Proteins
  • Trans-Activators