DOI: 10.57760/sciencedb.07493
title: NLRP6 self-assembles into a linear molecular platform following LPS binding and ATP stimulation
title: NLRP6 self-assembles into a linear molecular platform following LPS binding and ATP stimulation

description: Expression and purification of full-length NLRP6 monomer. Full-length NLRP6 fused with N-terminal His-tag followed by a maltose binding protein (MBP) tag were expressed in E. coli, and purifed by Ni-NTA (QIAGEN) afnity chromatography followed by Amylose afnity chromatography (NEB). Afer HRV3C digestion, high purity NLRP6 protein preparations in the putative ligand binding oligomeric state were obtained. Using protein solubility test38 and analytical gel fltration, an appropriate bufer condition (20 mM MES pH 6.5, 0.5M NaCl, 5% glycerol and with 0.2‒0.5M Arginine) was identifed out to keep NLRP6 in a monomeric state.Screening of NLRP6 potential ligands. To identify the potential ligand of NLRP6, a panel of microbial components including PS(Ra)26 were selected to be incubated with NLRP6 monomer in 20:1 molar ratio, and then loading to a Superdex 200 10/300 column (GE Healthcare).Electron microscopy and image processing. Preparation of negatively stained samples and image acquisition were as described elsewhere39. Te image processing was using EMAN and EMAN2.SPR experiment. In order to investigate the interaction between LPS and NLRP6, a kinetics assay was performed at 298K using a Surface Plasmon Resonance (SPR) Biacore 3000 machine (GE Healthcare, Uppsala,Sweden). Kinetic profling was performed using the single cycle kinetics method25. Data were analyzed using Biacore 3000 and ft to a 1:1 Langmuirbinding model.Live cell imaging. Live cell imaging was performed using a DeltaVision live cell imaging system (Applied Precision) equipped with an Olympus IX-71 inverted microscope and a 100×, 1.40N.A. oil objective. Images were captured with 100ms exposure times in 30min intervals by a CoolSnap HQ2 CCD camera, and diferent Z sections were projected by SofWorx suite.Data availabilityWe declare that all the data supporting the findings of this study are available within the paper and the Supplementary Information fles.
description: Expression and purification of full-length NLRP6 monomer. Full-length NLRP6 fused with N-terminal His-tag followed by a maltose binding protein (MBP) tag were expressed in E. coli, and purifed by Ni-NTA (QIAGEN) afnity chromatography followed by Amylose afnity chromatography (NEB). Afer HRV3C digestion, high purity NLRP6 protein preparations in the putative ligand binding oligomeric state were obtained. Using protein solubility test38 and analytical gel fltration, an appropriate bufer condition (20 mM MES pH 6.5, 0.5M NaCl, 5% glycerol and with 0.2‒0.5M Arginine) was identifed out to keep NLRP6 in a monomeric state.Screening of NLRP6 potential ligands. To identify the potential ligand of NLRP6, a panel of microbial components including PS(Ra)26 were selected to be incubated with NLRP6 monomer in 20:1 molar ratio, and then loading to a Superdex 200 10/300 column (GE Healthcare).Electron microscopy and image processing. Preparation of negatively stained samples and image acquisition were as described elsewhere39. Te image processing was using EMAN and EMAN2.SPR experiment. In order to investigate the interaction between LPS and NLRP6, a kinetics assay was performed at 298K using a Surface Plasmon Resonance (SPR) Biacore 3000 machine (GE Healthcare, Uppsala,Sweden). Kinetic profling was performed using the single cycle kinetics method25. Data were analyzed using Biacore 3000 and ft to a 1:1 Langmuirbinding model.Live cell imaging. Live cell imaging was performed using a DeltaVision live cell imaging system (Applied Precision) equipped with an Olympus IX-71 inverted microscope and a 100×, 1.40N.A. oil objective. Images were captured with 100ms exposure times in 30min intervals by a CoolSnap HQ2 CCD camera, and diferent Z sections were projected by SofWorx suite.Data availabilityWe declare that all the data supporting the findings of this study are available within the paper and the Supplementary Information fles.