CqsS In vitro Kinase Assay Measured in Biochemical System Using Imaging - 2132-10_Agonist_Dose_DryPowder_Activity
In order to investigate how CAI-1 binding regulates quorum sensing signaling events, we used a biochemical approach to reconstitute the CqsS->LuxU->LuxO circuit in vitro. We cloned and over-expressed the CqsS receptor in recombinant E. coli, and examined in vitro auto- phosphorylation of CqsS using inverted membrane vesicles and [gamma-32P] ATP. In this assay, wild type CqsS exhibits more ..
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Keywords: kinase, CqsS, quorum, cholera, in vitro
In order to investigate how CAI-1 binding regulates quorum sensing signaling events, we used a biochemical approach to reconstitute the CqsS->LuxU->LuxO circuit in vitro. We cloned and over-expressed the CqsS receptor in recombinant E. coli, and examined in vitro auto- phosphorylation of CqsS using inverted membrane vesicles and [gamma-32P] ATP. In this assay, wild type CqsS exhibits auto-phosphorylation similar to what happens in cells. Compounds were tested for their ability to inhibit CqsS auto-phosphorylation. Reaction products are terminated and run out by SDS-PAGE. Gels are dried and exposed to a phosphoscreen. From epistatic studies in a number of different bacterial strains, we know that our lead compounds act upon CqsS or require CqsS for activity because they are inactive in the absence of CqsS. It is likely that agonism occurs by preventing the auto-phosphorylation or preventing a change in the protein that results from auto-phosphorylation. A probe candidate that inhibits auto-phosphorylation is acceptable but so is a compound that works by some other method, such as allosteric modification.
Expected Outcome: A decrease in phosphorylation will coincide with a less radiolabel incorporation in the gel band (corresponding with CqsS) and suggest that a compound is inhibiting cqsS auto-phosphorylation. No activity in this assay is also acceptable if the compound works some other mechanism.
Preparation of inverted membranes
E. coli BL21 (DE3) harboring plasmids encoding wild type CqsS were grown with shaking in LB with 100 mug/mL kanamycin at 37 degrees C. Overnight bacterial cultures were diluted 1:100 into fresh LB medium with kanamycin. After growth with aeration for 3 h at 37 degrees C, protein production was induced with 1 mM IPTG. The cultures were shifted to room temperature and grown for an additional 5 h with shaking. Cells were harvested by centrifugation, resuspended in lysis buffer (50 mM Tris pH 8.0, 200 mM NaCl, 5 mM beta-mercaptoethanol, 5 mM MgCl2 and 20 mM imidazole), and lysed under 15,000 psi. Cell lysates were centrifuged at 9,300 x g for 30 min and the cleared fluids were harvested and subjected to ultra-centrifugation at 180,000 x g for 1 h. Membrane pellets were resuspended in kinase buffer (50 mM Tris pH 8.0, 100 mM KCl, 5 mM MgCl, and 10% (v/v) glycerol) and quantified by SDS-PAGE and Western blot using CqsS purified in detergent as the standard.
Phosphorylation assays were performed with inverted membranes containing 2 muM wild type CqsS protein. In assays containing LuxU and/or LuxO, LuxU and LuxO were supplied at 10 muM. Reactions were carried out in phosphorylation buffer (50 mM Tris pH 8.0, 100 mM KCl, 5 mM MgCl, and 10% (v/v) glycerol), and were initiated with the addition of 100 muM ATP and 2 muCi [gamma-32P]-ATP (from a stock of 3000 Ci/mmol: Perkin Elmer). For experiments with CAI-1 and analogs, compounds were added 10 min prior to the initiation of the reactions. For the reconstitution of the complete CAI-1/CqsS, LuxU, LuxO circuit, CAI-1 was supplied at 500 muM. Regulation of histidine phosphorylation by CAI-1 was assayed with 500 muM CAI-1. Agonism was tested at 100 muM. Antagonism was tested with 10 muM agonist and 100 muM. Reactions were incubated at room temperature and terminated with SDS-PAGE loading buffer. Reaction products were separated using SDS- PAGE. Gels were dried at 80 degrees C on filter paper under vacuum, exposed to a phosphoscreen overnight, and subsequently analyzed using a Typhoon 9400 scanner and ImageQuant software.
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