Journal: PLoS ONE
Article Title: Direct Binding of Ledipasvir to HCV NS5A: Mechanism of Resistance to an HCV Antiviral Agent
Figure Lengend Snippet: Characterization of purified NS5A. (A) Coomassie staining of purified proteins. Recombinant His-tagged proteins were purified as described in Materials and Methods. 1 μg of NS5A-6HIS and NS5A-Y93H-6HIS were subjected to SDS-PAGE and the protein bands were visualized by Coomassie Blue stain. Molecular weights for protein standards are indicated. (B) Phosphorylation sites determined by mass spectrometry of purified NS5A-6HIS and NS5A-Y93H-6HIS. Phosphorylation sites identified in NS5A-6HIS are shaded green; sites identified in NS5A-Y93H-6HIS are shaded red; and sites identified in both proteins are shaded cyan. (C) Analytical ultracentrifugation analysis of NS5A-6HIS, NS5A-Y93H-6HIS, and NS5A-domain 1. For NS5A-6HIS and NS5A-Y93H-6HIS, analytical ultracentrifugation was performed in 25 mM Tris pH 7.5, 150 mM NaCl, 0.01% NaN 3 , 0.5 mM TCEP, 2 μg/ml Leupeptin, 0.02% C12E8, and 21.1% (v/v) D 2 O. Sedimentation velocity analysis was performed at 42,000 rpm for two protein concentrations (3.3 and 6.6 μM). For NS5A-domain 1, analytical ultracentrifugation was performed in 25 mM Tris pH 8.0, 250 mM NaCl, 10% glycerol, and 0.5% DMSO. Sedimentation velocity analysis was performed at 48,000 rpm for 4 protein concentrations (7.5, 12, 15, 30 μM). Representative traces are shown for each protein. (D) Circular dichroism spectroscopy of NS5A-6HIS and NS5A-Y93H-6HIS. Circular dichroism measurements of NS5A-6HIS or NS5A-Y93H-6HIS (10 μM) were carried out at 20°C. The average of 3 measurements is shown.
Article Snippet: The PCR fragment was digested with Xba1 and EcoRV and ligated into pcDNA3.1(-) (Invitrogen). pcDNA3-NS5A-Y93H was generated by site-directed mutagenesis (Stratagene). pTK-NS5A was constructed by digesting pcDNA3-NS5A with Xba1 and EcoRV to isolate a 1.5 kb fragment encoding NS5A.
Techniques: Purification, Staining, Recombinant, SDS Page, Mass Spectrometry, Sedimentation, Spectroscopy