gst fusion protein purification column  (Beyotime)

Journal: Nature Communications

Article Title: SHANK3 depletion leads to ERK signalling overdose and cell death in KRAS-mutant cancers

doi: 10.1038/s41467-024-52326-1

Figure Lengend Snippet: a Schematic of the SHANK3 protein domains and crystal structure model of the SHANK3 SPN domain in complex with active KRAS. The zoom-in shows the critical interacting amino acids. Modified from our previous publication . SPN, Shank/ProSAP N-terminal domain; ARR, ankyrin repeat domain; SH3, Src homology 3 domain; PDZ, PSD-95/Discs large/ZO-1 domain; PP, proline-rich region; SAM, sterile alpha motif domain. b MST binding curve for the indicated proteins. The affinity curve and Kd -value are obtained from triplicate measurements (mean ± s.e.m.; representative of two independent experiments). c , d ITC titration and isotherms for interaction between the indicated proteins. Solid line in c indicates fitting to the single-site-bin ding model at 25 °C with 350 µM of KRASQ61H and 20 µM of SPN-ARR (graphs are a representative of three technical replicates; one independent experiment). e Immunoprecipitation (IP) in HEK293 cells co-expressing mRFP-SHANK3 WT and GFP-KRASG12V using mRFP-trap beads. A representative western blot is shown (three independent experiments). f IP in HEK293 cells co-expressing the GFP-tagged SHANK3 SPN domain (WT or RAS-binding-deficient mutant, R12E/K22D) and KRASG12V-dsRed using GFP-trap beads. A representative western blot is shown (three independent experiments). g FRET assay between GFP-tagged SHANK3 SPN domain (WT or R12E/K22D; FRET donor) and mCherry-KRASG12V (FRET acceptor) in HEK293 cells. Quantification of relative FRET efficiency, normalised to mCherry control vector (see methods). Individual data points and the population average of each biological replicate are shown [mean ± s.d.; three independent experiments; unpaired two-tailed Student’s t -test with Welch’s correction; 190 (mCherry + WT SPN), 227 (mCherry-KRASG12V + WT SPN), 108 (mCherry + R12E/K22D SPN), 153 (mCherry-KRASG12V + R12E/K22D SPN) individual data points analysed]. h , i Rescue of cell viability after SHANK3 silencing. Quantification of viable GFP- or mCherry-positive MIA PaCa-2 cells expressing either full-length GFP-SHANK3 WT or mutant R12E/K22D ( h ), or GFP/mCherry-tagged SHANK3 SPN domain ( i ) after endogenous SHANK3 silencing (36 h). Shown are individual data points [mean ± s.d., n = 4 ( h ) and 3 ( i ) independent experiments ( i , each replicate is shown in a different colour); one-way ANOVA with Holm-Sidak’s multiple comparison test]. Source data are provided as a Source Data file.

Article Snippet: The GST KRASG12V fusion protein was purified with Protino Glutathione Agarose 4B (Macherey-Nagel, Düren, Germany) and GST was cleaved by Tobacco Etch Virus (TEV) protease (Invitrogen, Life Technologies, Carlsbad, CA) at 4 °C for 16 h. The TEV protease cleavage extended KRASG12V construct in the N-terminal by four amino acid residues, G, A, M and G. The proteins were further purified by size exclusion chromatography (SEC) with a HiLoad 26/60 Superdex 200 pg column (GE Healthcare, Chicago, IL) in SEC buffer (50 mM Tris, pH 7.3, 300 mM NaCl, 1 mM DTT, 0.1% CHAPS) using an ÄKTA pure chromatography system (GE Healthcare).

Techniques: Modification, Sterility, Binding Assay, Titration, Immunoprecipitation, Expressing, Western Blot, Mutagenesis, Control, Plasmid Preparation, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: SHANK3 depletion leads to ERK signalling overdose and cell death in KRAS-mutant cancers

doi: 10.1038/s41467-024-52326-1

Figure Lengend Snippet: a Representative images of GFP-SHANK3 and mCherry-KRASG12V localisation in A549 cells (maximum projections shown; one experiment with this cell line). Insets and yellow arrows indicate colocalization of GFP-SHANK3 with mCherry-KRASG12V at membrane protrusions. b KRAS–SHANK3 SPN-ARR in an open conformation modelled by aligning the RBD and SPN domains of RAF and SHANK3, respectively, on a membrane composed of POPC (1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine)/ Phosphatidylinositol 4,5-bisphosphate/ Cholesterol. c Structural alignment between KRAS–SHANK3 SPN-ARR (model) and nanodisc-bound KRAS–RAF complex (PDB:6PTW). d Analysis of RAF-RBD–KRAS binding in the presence of the SHANK3 SPN domain using the depicted pulldown assay. Samples were resolved on SDS-PAGE gel and stained with Coomassie. A representative gel is shown (three independent experiments). e Quantification of relative FRET efficiency between GFP-KRASG12V (FRET donor) and mRFP-RAF-RBD (FRET acceptor) in siCTRL or siSHANK3 (smartpool SHANK3 siRNA) HEK293 cells. Shown are the individual data points [mean ± s.d., n = 79 (siCTRL) or 87 (siSHANK3) from three independent experiments. Unpaired two-tailed Student’s t -test with Welch’s correction]. f A representative immunoblot and quantification of ERK activation levels (phospho-ERK1/2 (Thr202/Y204) / total ERK relative to loading) in HCT-116 cells expressing GFP-SHANK3 SPN WT or GFP-SHANK3 SPN R12E/K22D (data represent the individual values; mean ± s.d.; mean of control is set to 1.0 by definition; three independent experiments; Kruskal-Wallis one-way ANOVA and Dunn’s post hoc test). g Representative confocal images (middle plane) and quantification of nuclear ERK (indicating ERK activity) in MIA PaCa-2 cells. Yellow arrowheads point to representative nuclei. N/C, nuclear to cytoplasmic ratio. Shown are the individual data points and the population average of each biological replicate (mean ± s.d.; three independent experiments; one-way ANOVA with Holm-Sidak’s multiple comparison test). h Representative images and quantification of tumour growth of HCT-116 cells, transiently expressing GFP-SHANK3 SPN WT or GFP-SHANK3 SPN R12E/K22D, on CAMs. Tumours are delimited by the yellow circles [mean ± s.d.; n = 21 (GFP, SPN WT) or 19 (SPN R12E/K22D) tumours from two independent experiments; Kruskal-Wallis one-way ANOVA and Dunn’s post hoc test]. Source data are provided as a Source Data file.

Article Snippet: The GST KRASG12V fusion protein was purified with Protino Glutathione Agarose 4B (Macherey-Nagel, Düren, Germany) and GST was cleaved by Tobacco Etch Virus (TEV) protease (Invitrogen, Life Technologies, Carlsbad, CA) at 4 °C for 16 h. The TEV protease cleavage extended KRASG12V construct in the N-terminal by four amino acid residues, G, A, M and G. The proteins were further purified by size exclusion chromatography (SEC) with a HiLoad 26/60 Superdex 200 pg column (GE Healthcare, Chicago, IL) in SEC buffer (50 mM Tris, pH 7.3, 300 mM NaCl, 1 mM DTT, 0.1% CHAPS) using an ÄKTA pure chromatography system (GE Healthcare).

Techniques: Membrane, Binding Assay, SDS Page, Staining, Two Tailed Test, Western Blot, Activation Assay, Expressing, Control, Activity Assay, Comparison