원문정보
초록
영어
Quorum-sensing (QS) is involved in various cellular events such as cell density control, secondary metabolite production, cell differentiation, biofilm formation, and virulence. The quorum sensing system of Pseudomonas aeruginosa is currently the most intensively studied, because this bacterium is an opportunistic human pathogen responsible for the death of thousands of cystic fibrosis sufferers and many other immunocompromised individuals each year. Quorum sensing inhibitors can attenuate the pathogenicity of P. aeruginosa. Here we report the production and characterization of LasR protein, a transcriptional activator in P. aeruginosa, from a recombinant E. coli BL21. LasR proteins, either His-tagged or GST-tagged, could be produced as soluble proteins, only when a proper signaling molecule, acyl homoserine lactone (AHL), was added in the culture medium. It was confirmed that the soluble LasR proteins produced in the presence of AHL had a binding affinity to the promoter OP1 (lasB elastase
promoter). Without added AHL, most proteins were produced as inclusion body in the cell. Some soluble LasR proteins could be obtained from AHL-free culture medium but they did not have binding affinity to OP1 sequence. Based on gel retardation method, we tried to develop a biochemical method which directly measures the effect of various chemicals on the interaction between transcription activator protein and signaling molecules and/or the binding of the protein-AHL complex to the corresponding DNA sequence. However, active LasR was always obtained in the form of the protein-AHL complex; in vitro, the conversion of LasR-AHL complex to apo-LasR (AHL-free form protein) or the conversion of apo-LasR to active LasR-AHL complex was not possible. In addition, in vitro supplementation of various AHLs to LasR-AHL complex did not
affect the binding affinity of the purified protein-AHL complex to the OP1 DNA sequences. These results suggest that the incorporation of AHL in LasR protein is not reversible because it probably occurs before the protein is fully folded during its production. The development of the biochemical assay with LasR will be an extremely challenging task which requires extensive studies on the structure of LasR and its folding processes.