Escherichia coli BL21 star (DE3) cells (Invitrogen) were transformed with histidine-tagged pET151/D-Topo FMRP full-length, KH1, KH2 or RGG box constructs (6 (link)). The cells were grown in LB medium with ampicillin (100 mg/ml) and induced for 4 h at 20°C by addition of 1mM isopropyl-β-D-thiogalacto-pyranoside when the cultures reached an OD of 0.4 at 600 nm. Cells were then harvested and resuspended in lysis buffer [25 mM Tris–HCl pH 7.6, 300 mM KCl or LiCl, 1 mM EDTA, 1 mM dithiothreitol (DTT) , 20% glycerol, 5% NP-40, 0.5M urea, complete protease inhibitor cocktail (Roche), 1 mM PMSF] sonicated for 5 min and centrifuged at 15 000 r.p.m. for 30 min at 4°C. The supernatant was incubated with Ni-NTA Agarose beads (Qiagen) for 2 h at 4°C by agitation. Beads were washed four times at 4°C with washing buffer (25 mM Tris–HCl, pH 7.6, 300 mM KCl, 1 mM DTT, 0.5M urea, 20% glycerol, 20 mM imidazole). Fusion proteins were eluted from the beads with elution buffer (25 mM Tris–HCl pH 7.6, 50 mM KCl, 1 mM EDTA, 1 mM DTT, 10% glycerol, 0.1% Triton, 0.5M urea, 250 mM imidazole) for 30 min on ice.