Figure 1

Expression and purification of N-terminally truncated forms of NS5A. (a) Schematic of the structure of NS5A and the expressed truncated forms showing the locations of the N-terminal amphipathic helix and the three domains. (b) Anti-NS5A western blot analysis and Coomassie staining of the purification of NS5A(Δ32) (left) and NS5A(Δ250) (right). Lanes: 1; uninduced lysate, 2; induced lysate, 3; lysate clarified by centrifugation and applied to the Ni-NTA column, 4; flow-through, 5–8; washes, 9; 200 mM imidazole elution, 10; gel filtration eluate, 11; Coomassie stain of gel filtration eluate. (c) Slow crystallization mass spectrometry of NS5A(Δ32) (left) and NS5A(Δ250) (right). The peak at ~69 kDa on the left represents the E. coli chaperone protein DnaK. The measured molecular masses correspond closely to the predicted values: NS5A(Δ32): 47,222 Da, and NS5A(Δ250): 26,500 Da. (d) Interaction of truncated NS5A forms with the Fyn SH3 domain. Purified NS5A(Δ32) (left) and NS5A(Δ250) (right) were subjected to GST pulldown analysis using GA-beads alone (lanes 2), GST (lanes 3) or GST-FynSH3 (lanes 4). Samples were blotted for NS5A, lanes 1 show 20% of input protein, lanes 2–4 show bound protein eluted by competition with 20 mM reduced glutathione. The lower panel shows a Coomassie stained SDS-PAGE of the purified GST-FynSH3 domain fusion proteins.