Dataset Information

Target deconvolution in Plasmodium falciparum using pulldowns

ABSTRACT: Parasite lysates were centrifuged (20,000 g, 4°C, 15 min). Beads (blank and with linker attached) were washed 3× with water then twice with lysis buffer. The lysate (~2 mg total protein) was first incubated with 2 mg blank beads for 30 min at 4°C with rotating agitation. The lysate was divided into two then incubated with either 1% DMSO or 100 µM DDD01510706 (competitor) for 30 min at 4°C with agitation. Finally, lysates were incubated with 2 mg of compound-bound beads for 1 h at 4°C with agitation. The beads were then washed 3× with wash buffer (0.8% (w/v) octyl β-D-glucopyranoside, 50 mM Tris pH 8.0, 5 mM EDTA, 1 mg/mL BSA) and 2× Tris-buffered saline (TBS; 50 mM Tris-Cl pH 7.5, 150 mM NaCl). Samples were run 1.5 cm into a Bis-Tris 10% (w/v) acrylamide gel and stained with Coomassie quick reagent for 30 min. The entire gel bands were removed and subjected to in-gel reduction with 10 mM dithiothreitol, alkylation with 50 mM iodoacetamide and digestion with 12.5 μg/mL trypsin (Pierce) for >16 h at 37°C. Recovered tryptic peptides were then vacuum dried prior to analysis

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Plasmodium Falciparum (isolate 3d7)

SUBMITTER: Victoriano Corpas-Lopez

LAB HEAD: Susan Wyllie

PROVIDER: PXD033740 | Pride | 2022-10-15

REPOSITORIES: Pride

ACCESS DATA

Publications

Toolkit of Approaches To Support Target-Focused Drug Discovery for <i>Plasmodium falciparum</i> Lysyl tRNA Synthetase.

Milne Rachel R Wiedemar Natalie N Corpas-Lopez Victoriano V Moynihan Eoin E Wall Richard J RJ Dawson Alice A Robinson David A DA Shepherd Sharon M SM Smith Robert J RJ Hallyburton Irene I Post John M JM Dowers Karen K Torrie Leah S LS Gilbert Ian H IH Baragaña Beatriz B Patterson Stephen S Wyllie Susan S

ACS infectious diseases 20220829 9

There is a pressing need for new medicines to prevent and treat malaria. Most antimalarial drug discovery is reliant upon phenotypic screening. However, with the development of improved target validation strategies, target-focused approaches are now being utilized. Here, we describe the development of a toolkit to support the therapeutic exploitation of a promising target, lysyl tRNA synthetase (<i>Pf</i>KRS). The toolkit includes resistant mutants to probe resistance mechanisms and on-target en ...[more]

PMID: 36037410

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Project description:Glioblastoma stem cells (TS-543) were harvested and cross-linked with 1% formaldehyde for 10 mins at room temperature. The cells were lysed using SDS Lysis buffer for ChIP (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8). The lysate was then sonicated for 25 cycles at 30% amplitude (15 s ON and 45 s OFF). The sonicated samples were then diluted in ChIP dilution buffer (0.01% SDS, 1% Triton-X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl pH 8, 167 mM NaCl) and used for the immunoprecipitation with H3K27ac (Abcam, Ab4729), H2AZ (Active motive, cat#39113) and H3K27ac (Abcam cat#ab8580). After an over-night incubation with antibody, the bound DNA was washed sequentially with low salt wash buffer (0.1% SDS, 1% Triton X 100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 150 mM NaCl), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 500 mM NaCl), LiCl wash buffer (0.25 M LiCl, 1% NP40, 1%deoxycholate, 1 mM EDTA, 10 mM Tris–HCl pH 8) and TE wash buffer (10 mM Tris–HCl pH 8, 1 mM EDTA) to remove non-specific sequences and eluted in the elution buffer (84 mg NaHCO3, 1 ml 10% SDS, 9 ml H2O). Then the samples were reverse cross-linked using NaCl at 65°C overnight. The eluted DNA was purified and used for library preparation. Library preparation was performed as previously described as described in Bowman et al. BMC Genomics 2013. Briefly, end repair with NEBNext End Repair enzyme (NEB, E6050) and clean-up with 2.4x volume AMPure XP beads (Beckman Coulter, A63880). A-tailing with Klenow Fragment (NEB, M0212) and purified with 2.4x volume AMPure XP beads. Adapter ligation reactions contained annealed universal adapter and T4 rapid ligase (NEB, B0202) and clean-up with 1.8x volume AMPure XP beads. Chromatin was amplified using KAPA Real-time Library Amplification Kit (KAPABIOSYSTEMS, KK2701) with universal primer and barcoded primer. Amplified chromatin was purified with QIAquick Gel Extraction Kit (Qiagen) and sequenced paired-end with Illumina NextSeq 500.

Dataset Information

Target deconvolution in Plasmodium falciparum using pulldowns

Publications

Toolkit of Approaches To Support Target-Focused Drug Discovery for <i>Plasmodium falciparum</i> Lysyl tRNA Synthetase.

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