Purification of Mutant Ran GTPase

N-Terminal strep-6xHis-TEV mTagBFP2 RanQ69L was cloned into the pST50 vector via Gibson Assembly (New England Biolabs). The plasmid was transformed into E. coli Rosetta2 cells (EMD Millipore) for protein expression. Cells (4 L) were grown until OD₆₀₀ = 0.8 at 37 °C in LB media, and then protein expression was induced using 500 mM IPTG for 18 hours at 16 °C before cells were pelleted. RanQ69L was purified in the following manner:^{24 (link),25 (link)} we lysed cells by using an Emulsiflex french press (Avestin) in binding buffer (100 mM Tris–HCl, 450 mM NaCl, 1 mM MgCl₂, 1 mM EDTA, 2.5 mM PMSF, 6 mM BME, pH 8.75). We centrifuged the lysate at 20 000 × g and loaded the supernatant onto a StrepTrap HP column (GE Healthcare, 5 ml). Protein was eluted in binding buffer with 2.5 mM d-desthiobiotin, and dialyzed overnight into CSF-XB buffer (10 mM HEPES, 100 mM KCl, 1 mM MgCl₂, 5 mM EGTA, 10% sucrose w/v, pH 7.7). We included 200 μM GTP in lysis and elution buffers and obtained about 0.5 ml of 200 μM RanQ69L.