Fig. 1: Purification of a recombinant CCDC6-RET fusion product from Sf9 insect cells.
From: The oncogenic CCDC6-RET fusion protein is a dual ATP- and ADP-dependent kinase
a Schematic diagram of the CCDC6-RET fusion product depicting functional domains with the indicated phospho-sites and aa number. b In tandem immobilized metal affinity (Ni2+), glutathione-conjugated gravity flow chromatography and in-gel 3C-protease digestion. Indicated fractions were run on an SDS-PAGE and stained with Coomassie: 1–4 elution fractions with 100% buffer B, 5 recombinant protein bound to the glutathione conjugated resin before proteolytic cleavage, 6–7 elution fractions after cleavage with 3C-PreScission protease (GST-tagged, see lane 8), 8 glutathione conjugated resin after proteolytic cleavage. Size exclusion chromatography (sec) using a Superdex 200 3.2/300 column. Indicated fractions were run on an SDS-PAGE and stained with Coomassie (lower panel). Data are representative of n ≥ 10 experiments. c Absorbance spectra (260–310 nm) with max at 280 nm and indicated (260/280 nm) ratio. d Mass photometry profiles (counts vs mass in kDa) of CCDC6-RET at different concentrations in color code: 50 nM (magenta), 25 nM (blue), 10 nM (green), 5 nM (orange). e DSF profile (IF) of CCDC6-RET (2 μM) in the apo state and bound to BLU-667 (10 μM) and Ponatinib (10 μM). f Ti (°C) and ΔTi (°C) values from (f). Data (mean ± SEM) of three experiments, n = 3. g WB of samples from a time-course autophosphorylation experiment with CCDC6-RET (1 μM) in the presence of ATP (1 mM) and MgCl2 (2 mM) for 0–30 min using a total anti-phospho-tyrosine antibody. The total amount of protein was visualized by Coomassie staining. Data are representative of n ≥ 10 experiments. h Enzymatic assay performed with CCDC6-RET (0.25 μM) incubated with increasing concentrations of ATP at a fixed concentration (1.35 mM) of a c-Abl-derived peptide (EAIYAAPFAKKK). Data represent the mean ± SEM of three experiments in duplicate (n = 6). Enzymatic activity (ODs−1 × 10−2) versus ATP concentration and KM values are depicted. i Consumption curves OD (λ 243 nm) versus time (in seconds) at increasing concentrations of ATP (0–2.5 mM) of a representative enzymatic assay from (h). Source data are provided as a Source data file.
