Development and Characterization of LasR Immobilized Monolithic Column for Screening Active Ingredients as Quorum...

The enzyme/protein immobilized monolithic capillary combined with liquid chromatography-mass spectrometry is an efficient screening strategy for the corresponding agonist/antagonist. LasR is the potential therapeutic target since it plays a vital role in the colonization and invasion of Pseudomonas aeruginosa (P. aeruginosa). Therefore, reagents that inhibit LasR may be effective against P. aeruginosa. To screen and find LasR inhibitors rapidly, a LasR-immobilized monolithic capillary column was prepared and characterized.

Firstly, the recombinant LasR protein was prepared in E. coli. Then, the LasR protein was immobilized to the surface of poly (glycidyl methacrylate-co-poly(ethylene glycol)diacrylate)-ethylenediamine monolithic column. The affinity and stability of prepared column was also evaluated. Furthermore, the prepared column was applied to fishing LasR inhibitor in Scutellaria baicalensis Georgi extract. The interaction of the screening compound to LasR was confirmed through molecular docking.

The recombinant active LasR protein was prepared in E. coli. After purification and validation, a comparative ligand fishing monolithic column was prepared through immobilizing LasR to the surface of the poly (glycidyl methacrylate-co-poly(ethylene glycol)diacrylate)-ethylenediamine through amidation reaction. The LasR was successfully immobilized to the monolithic column characterizing by Fourier transform infrared spectroscopy and scanning electron microscopy. The activity of immobilized LasR was reserved as it has affinity to the nature ligand 3-oxo-C₁₂-HSL and stablied within 24 h at 4 °C. In the Scutellaria baicalensis Georgi extract, baicalein was screened as a potential LasR inhibitor. The molecular docking results and the in vivo evaluation confirmed the activity of baicalein.

The proposed LasR immobilized monolithic column is a viable strategy in screening LasR inhibitors. It can be considered as a possible alternative to traditional methods for screening LasR inhibitors as drug candidates against P. aeruginosa.