Glutathione sepharose

GST-tagged proteins were all produced in BL21 E. coli from pGEX-based plasmids, and the protein expression was induced with 0.1 mM isopropyl-β-d-thiogalactopyranoside at 22°C for 5 h when fresh bacterial cultures grew to A600 of 0.6–0.8 at 37°C. From induced cultures, cells were harvested by centrifugation, washed with cold phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4), and lysed in PBS supplemented with 250 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 3 mM DTT, 0.5% Triton X-100, and 1 mg/ml lysozyme (lysis buffer) at 4°C for 30 min. On average, 3 ml of lysis buffer were used to lyse pelleted cells from a 100-ml culture. After cell lysates were sonicated and centrifuged at 13,000 × g at 4°C for 15 min, SNs were collected and mixed with 1/20 vol of glutathione sepharose (GenScript) on rotation at 4°C overnight. After beads were pelleted, they were washed three times with a wash buffer consisting of 50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5% Triton X-100, and 0.5% Tween-20 and rinsed three times with PBS. The average concentration of immobilized GST-tagged proteins ranged from 0.3 to 3 µg/µl. The beads were stored in PBS at 4°C for up to a week before being used for phosphorylation reactions.

GST-tagged human MNK1a and MNK1b were subcloned into the BamHI and NotI sites of pGEX-4T3 and expressed in Escherichia coli Rosetta cells. Proteins were purified with glutathione-Sepharose (GenScript, Piscataway, NJ) according to the manufacturer’s instructions. Briefly, expression was induced with 1 mM IPTG for 2 h at 25°C. Cells were suspended in buffer containing 5 mM sodium phosphate, 150 mM NaCl, 1 mM EDTA (pH 7.4), and 1 mg/mL lysozyme, and incubated on ice for 30 min. Afterward, 0.5% Triton X-100 was added and bacteria were subjected to sonication. Cell debris was removed by centrifugation, and the supernatant was incubated with glutathione-Sepharose equilibrated in the same buffer by rocking for 2 h at 4°C. Finally, after extensive washes with the same buffer, the proteins were eluted with 10 mM glutathione in 50 mM Tris-HCl (pH 8).
His-MNK1b, cloned as described in³¹ (link) was purified by affinity chromatography by ProAlt (Protein Alternatives S.L., Madrid, Spain).