Sample preparation for structural and ITC binding analysis

Rat SHANK3 (residues 1-348) was expressed using BL21 competent cells (Invitrogen) cultured in LB (Luria Broth) for crystallization and ITC, or M9 minimal medium for NMR analysis. Cells were grown at 37°C in LB media supplemented with antibiotics to an OD₆₀₀ of 0.7, cells were cooled to 18°C and induced using 300 μM IPTG (Isopropyl β-D-1-thiogalactopyranoside) for 16 h. Cells were pelleted by centrifugation and resuspended in 20 mM Na₂HPO₄ pH 7.4, 500 mM NaCl and 25 mM imidazole, cells were then treated with protease cocktail inhibitor VII (Calbiochem) and bovine deoxyribonuclease (1 mg/ml) before being lysed using a French Press. Proteins were purified using nickel-affinity chromatography with a linear gradient of lysis buffer containing 500 mM imidazole. SUMO tag was cleaved with recombinant His-tagged sumo protease and removed by a reverse pass on the Ni-NTA column. Following this step the SHANK3 was shown to have > 95% purity by SDS-PAGE chromatography. SHANK3 was then exchanged into 20 mM Tris pH 7.4, 150 mM NaCl, 2 mM DTT (dithiothreitol) and concentrated to 6 mg/ml for crystallization experiments. H-Ras was expressed using Rosetta pLacI (Invitrogen) cells and purified using nickel affinity chromatography as described for SHANK3. The GST tag was cleaved with recombinant his-tagged 3C protease and removed by a reverse pass on the Ni-NTA column. H-Ras required subsequent purification using anion exchange purification using a QFF column (GE healthcare) and was shown to be > 95% pure by SDS-PAGE chromatography. Rap1b was expressed in E. coli strain CK600K. Cultures were grown at 37°C to OD₅₉₅ of 0.8 and then induced with 200 μM IPTG at 18°C overnight. Protein was purified by ion exchange, followed by gel filtration, as described in ⁷⁵. Active GTP form of Ras and Rap1b was produced by nucleotide exchange as described in ⁷⁶ using non-hydrolysable GTP analogue GMPPCP. GDP form was generated by overnight incubation of GTPase in the presence of 50 fold molar excess of GTP in the presence of EDTA to remove magnesium, followed by the buffer exchange using PD10 column (GE healthcare). Completeness of the exchange was validated by NMR spectroscopy.