X-ray structure and activities of an essential Mononegavirales L-protein domain

Paesen, Guido C.; Collet, Axelle; Sallamand, Corinne; Debart, Françoise; Vasseur, Jean-Jacques; Canard, Bruno; Decroly, Etienne; Grimes, Jonathan M.

Purification. Seventy-two hours after infection of the suspension cultures with recombinant baculovirus, Sf21 cells were spun down (1,000g, 7 min, 22°C) and lysed in one-fourth volume of buffer L (1.5% triton X-100, 5% glycerol, 50 mM arginine, 300 mM KCl, 10 mM imidazole and 20 mM Tris (pH 8.0)). After clarification (10,000g, 25 min, 4°C), the lysate was incubated with benzonase (Novagen; to 0.2 U ml À 1 ) and NiNTA resin (Qiagen; 0.3 ml l À 1 culture) for 5 h at 4°C, with gentle shaking. The beads were transferred to a 10-ml column and washed with 3 Â 10 ml buffer W (20 mM Tris (pH 8.0), 1.5 M NaCl, 10 mM imidazole and 7.5% glycerol) and 1.5 ml of 0.1 M arginine (pH 8.0). Protein was eluted in 0.8 M arginine (pH 8.0). An equal volume of ice-cold 3 M AmSO 4 was added to the eluent, and the precipitated protein collected (10,000g, 10 min, 4°C). Protein pellets were stored at À 20°C. A similar lysis and purification protocol was used to obtain recombinant protein from HEK293T cells. BL21 Star cells were lysed using Bugbuster Protein Extraction Reagent (Novagen). Expression and purification were monitored by SDS-PAGE and western blotting. Blots were developed with a 1/10,000 dilution of an anti-histidine tag antibody (Penta Á His Antibody, Qiagen, catalogue number 34660). Supplementary Fig. 1 shows purified protein following SDS-PAGE under reducing and non-reducing conditions. An uncropped western blot showing the unreduced, insect-cell-expressed protein next to a BenchMark (Invitrogen) molecular weight marker is also shown.