Amicon ultra centrifugation filter devices

All recombinant proteins were expressed as GST fusions. Plasmids were transformed into BL21 (DE3) CodonPlus-competent Escherichia coli (Stratagene), and expression was induced with 0.5 mm isopropyl 1-thio-β-d-galactopyranoside at 18 °C overnight. Cell pellets were resuspended in PBS containing 0.5 mm tris(2-carboxyethyl)phosphine and 5% glycerol and lysed by sonication. GST fusion proteins were affinity-purified from soluble bacterial lysates by using glutathione–Sepharose (GE Healthcare) according to standard manufacturer's protocols and cleaved off GST while bound to the beads by 2-h incubation at 30 °C with PreScission protease (GE Healthcare). Soluble recombinant proteins were either concentrated with Amicon Ultra centrifugation filter devices (Millipore) and stored as 5–10 mg/ml stocks at −80 °C until required (Smurf2), or the concentration was adjusted to about 0.5–1 mg/ml (polymerization-competent proteins) and stored at −80 °C.

Expression and purification of recombinant proteins were done essentially as described [16 ]. Briefly, plasmids were transformed into BL21 (DE3) CodonPlus-competent Escherichia coli (Stratagene). Protein expression was induced with 0.5 mM isopropyl beta-d-thiogalactoside (IPTG) at 30°C for 3 h (individual WW domains) or 18°C overnight (full-length WWP2). Cell pellets were resuspended in PBS containing 1 mM TCEP and lysed by sonication. GST fusion proteins were affinity-purified from bacterial lysates by using glutathione–sepharose (GE Healthcare) according to standard manufacturer's protocols. After elution, recombinant proteins were immediately rebuffered in resuspension buffer containing 5–10% glycerol using PD-10 desalting columns (GE Healthcare), concentrated with Amicon Ultra centrifugation filter devices (Millipore) and stored as 5–10 mg ml⁻¹ stocks at –80°C until required.