Expression and Purification of Recombinant GST-Tagged Proteins

The cDNA of human full-length SMN and truncations were inserted in pGEX-6P-1 (GE Healthcare) using BamHI and XhoI. Aromatic cage mutants were generated by site-directed mutagenesis using Pfu Turbo (Stratagene) followed by DpnI (NEB) digestion. Constructs were sequence-verified (GATC Biotech AG or Biofidal) and transformed into BL21 DE3 cells (Stratagene). BL21 cells were grown overnight with ampicillin selection at 37°C with agitation. The following day, cultures were scaled up in 250 ml LB (Sigma-Aldrich) and grown at 37°C until OD₆₀₀ ∼0.6. Then, expression of recombinant GST proteins was induced with 0.2 mM IPTG for 2.5–3 h at 37°C. Cells were harvested by centrifugation and resuspended in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.05% NP-40, supplemented Complete EDTA-free [Roche]). After a brief sonication, lysates were cleared by centrifugation and incubated with glutathione–sepharose (GE Healthcare) at 4°C on a tumbler wheel. After extensive washing, GST proteins were eluted with 10 mM reduced glutathione (Sigma-Aldrich) in 50 mM Tris, pH 8.0.