Alternative conformations of a major antigenic site on RSV F

Jones, Harrison G.; Battles, Michael B.; Lin, Chun-Chi; Bianchi, Siro; Corti, Davide; McLellan, Jason S.

Plasmid encoding prefusion-stabilized RSV F (DS-Cav1), subtype A DS-Cav1 with a K209Q substitution, or postfusion RSV F based on subtype A (strain A2) or subtype B (strain B9320) with a C-terminal 6x-or 8x-histidine tag and Strep-tag II was co-transfected with furin into FreeStyle 293-F cells (Invitrogen) at a 4:1 ratio to ensure full cleavage of prefusion RSV F. Proteins were purified from the media after six days using Ni-NTA Superflow resin (Qiagen) and Strep-Tactin resin (IBA). Tags were removed by digestion with thrombin protease, followed by gel filtration using a Superdex 200 16-600 column (GE Healthcare Biosciences). Prefusion RSV F protein used for SPR was produced in the same manner, except the tags were not removed prior to gel filtration. For crystallization, DS-Cav1 from strain A2 was expressed in the presence of kifunensine (5 μM), digested with 10% (w/w) Endo H overnight, mixed with a 2-fold or 1.5-fold molar excess of purified Fab for the AM22-RSV F and RSD5-GL-RSV F complexes, respectively, and the resulting complexes were purified by size exclusion chromatography (SEC) using the Superose 6 XK 16-70 column (GE Healthcare Biosciences) in a buffer consisting of 2 mM Tris pH 8.0, 200 mM NaCl, and 0.02% NaN 3 .