Biochemical characterization of recombinant influenza A polymerase heterotrimer complex: Endonuclease activity and evaluation of inhibitors
Fig 3
Biophysical characterization of purified influenza polymerase proteins.
(A) Analytical ultracentrifugation analysis of purified proteins. For PA, PA/PB1 dimer and PA/PB1/PB2 trimer, analytical ultracentrifugation was performed in a buffer containing 25 mM HEPES (pH 7.6), 300 mM NaCl, 5% glycerol, 0.5 mM TCEP, and 0.01% C12E8. Sedimentation velocity analysis was performed at 42,000 rpm. Representative traces for each protein are shown. (B-D) SEC-MALS analysis of purified PA (B) PA/PB1 (C), and PA/PB1/PB2 in the presence (black) or absence (green) of detergent during SEC purification (D). The normalized UV absorbance trace (280 nm) is plotted on the left axis. The horizontal gray lines correspond to the calculated mass by SEC-MALS (right axis).