Hiload superdex 75 column

Ubiquitin and the catalytic domain of Pol ι were expressed and purified in a similar manner. Briefly, plasmids encoding His Pol ι aa 1–419 and full-length ubiquitin were transformed into E. coli BL21(DE3) cells. Unlabeled proteins used for NMR experiments were expressed in 1 L of Luria broth (LB) media. The ¹⁵N-labeled sample of Pol ι was obtained by expressing proteins in M9 minimal medium supplemented with ¹⁵N-ammonium chloride as a sole source of nitrogen. Transformed cells were grown at 37 °C until OD₆₀₀ of 0.8–1.0. Protein expression was induced with 1 mM isopropyl 1-thio-β-ᴅ-galactopyranoside (IPTG) overnight at 20 °C. Cells were harvested by centrifugation, resuspended in a buffer containing 20 mM Tris pH 8.0, 200 mM NaCl, 10 mM imidazole and 1 mM PMSF, lysed by sonication, and centrifuged at 15,000 rpm for 30 min. The supernatant was filtered and applied to equilibrated HisPur^™ Cobalt resin (Thermo Scientific). Proteins were eluted in a buffer containing 20 mM Tris pH 8.0, 200 mM NaCl and 250 mM imidazole and then subjected to size-exclusion chromatography using a HiLoad Superdex 75 column (GE Healthcare) in a buffer containing 20 mM Tris pH 6.5, 100 mM NaCl and 10 mM β-mercaptoethanol.

Both S. cerevisiae 14–3–3 isoforms (Bmh1 and Bmh2) were expressed and purified as described previously [47] (link), [48] (link). The N-terminal 6 ×His-tag was removed using TEV protease, and final size-exclusion chromatography was performed in a HiLoad Superdex 75 column (GE Healthcare, Chicago, IL, USA). Bmh1 and Bmh2 were dialyzed overnight into a buffer containing 10 mM HEPES (pH 7.4) and 150 mM NaCl. Proteins at an initial concentration of 160 μM, followed by binary dilution series, were incubated for 1 h with 50 nM of FITC-labeled synthetic Trk1 peptide (FITC-RRCFpTLLFP), where pT denotes phosphothreonine (Pepscan Presto BV, Lelystad, Netherlands). The dilution series were performed in 384-well black low-volume flat-bottom plates (Corning, NY, USA) in a buffer containing 10 mM HEPES (pH 7.4), 150 mM NaCl, 0.1% (v/v) Tween 20% and 0.1% (w/v) BSA, using an epMotion P5073 pipetting robot (Eppendorf, Hamburg, Germany). Fluorescence polarization assay was measured using a CLARIOstar microplate reader (BMG Labtech, Thermo Fisher Scientific, Waltham, MA, USA) at 23 °C. The excitation and emission wavelengths were 482 nm and 530 nm, respectively. The K_D values were determined as the mean of four independent measurements.

For X-ray structure, recombinant proteins CWC22 and CWC27 were co-expressed in E. coli BL21 (DE3) grown in TB-medium at 37°C, as GST-3C-N-terminal His6 fusion or Sumo cleavable N-terminal His6 fusion proteins respectively. EIF4A3 was expressed as a Sumo cleavable N-terminal His6 fusion. Overexpression was induced at 18°C with 0.5 mM IPTG. Cells were lysed by sonication in 50 mM Na₂HPO₄ pH 7.5, 250 mM NaCl, 10 mM imidazole, 1 mM PMSF, and 25 mg/ml DNaseI, and the extract was cleared by centrifugation (4°C, 75 000 g, 30 min). In a first step, proteins were purified via a Ni²⁺-NTA affinity column (5 ml, GE healthcare). In order to remove N-terminal His6-tags, proteins were incubated with 3C or Senp2 proteases overnight at 4°C and dialyzed against 50 mM Na₂HPO₄ pH 7.5, 150 mM NaCl for subsequent heparin chromatography (5 ml Heparin Q sepharose, GE Healthcare). Protein complexes were isolated by size exclusion chromatography (SEC) after concentrating to 20–30 mg/ml in a buffer containing 10 mM HEPES pH 7.5, 150 mM NaCl and 1 mM DTT using a HiLoad Superdex 75 column (GE Healthcare). The complex was stored at 80°C in SEC buffer.