LsrK is one of the key components of the luxS-regulated (lsr) operon in Escherichia coli and plays an important role during the quorum-sensing (QS) process mediated by autoinducer-2 (AI-2). The AI-2 molecule is imported into the cell by the LsrACB transporter and is subsequently phosphorylated (to AI-2-P) by LsrK. AI-2-P binds to the repressor protein of the lsr operon (LsrR) and triggers various cellular responses related to QS by dissociating LsrR from the DNA. Although a large amount of purified LsrK is required for structural studies, recombinant GST-LsrK was mostly expressed in an insoluble form. To enhance the soluble expression of LsrK, an attempt was made to increase the expression of the cellular chaperone proteins that are well known to support proper protein folding. Transformed E. coli was cultured in high-salt LB medium and heat shock was applied prior to subsequent IPTG induction at 20°C. These procedures increased the yield of purified LsrK by about tenfold compared with standard IPTG induction at 20°C. The expressed LsrK was readily purified by GST-affinity chromatography. Crystals of LsrK were grown by the hanging-drop vapour-diffusion method. The X-ray diffraction data of the crystal were processed in a primitive hexagonal space group to 2.9 Å resolution.

Actinobacillus actinomycetemcomitans is a gram-negative, nonmotile coccobacillus bacterium that is associated with several human diseases, including endocarditis, meningitis, osteomyelitis, subcutaneous abscesses and periodontal diseases. A full-length Omp1-like protein gene from A. actinomycetemcomitans was cloned into a pQE30 vector and overexpressed in Escherichia coli BL21(DE3) cells. The protein revealed sequence homologies to Seventeen kilodalton proteins (Skp) from Pasteurella multocida and E. coli that have been characterized as periplasmic chaperones. This soluble Omp1-like protein was successfully purified to homogeneity for further folding and functional studies. The purity, identity, and conformation of the protein were determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis, matrix-assisted laser desorption ionization mass spectrometry, circular dichroism, fluorescence spectroscopic, and differential scanning calorimetric studies. We showed that the protein formed an oligomer larger than a tetramer. We found, further, that it is comprised of mostly α-helices and boasts high thermal stability.