Fig. 3

Expression of P9-GFP fusion protein. P9-GFP fusion protein was expressed alone or together with wild type P9 and the non-structural protein P12 in E. coli BL21(DE3) cells. Solid sucrose was added to the lysate (a–c) to obtain approximately 77% (w/v) sugar concentration for the equilibrium flotation centrifugation analysis. After flotation centrifugation, light scattering was detected under visible light (a). The positions of the P9 and P9-GFP vesicle fractions (V-P9 and V-P9-GFP, respectively), and the position of soluble GFP are indicated on the right. The proteins expressed are indicated above the tube images. BL21 indicates E. coli BL21(DE3) control cells (no phi6 protein expression). b, c Analysis of the flotation gradient fractions (top panel) by Western blotting using an anti-GFP antibody (bottom panel). The expressed proteins and the plasmids used are presented above. The position of the P9-GFP vesicle (V-P9-GFP) is indicated (c, top panel). The 40- and 30-kDa bands from MagicMark™ XP Western Protein standard (ThermoFisher Scientific) are indicated on the left (b and c; bottom panels). d Coomassie-stained SDS-PAGE gel of P9-GFP vesicles purified from E. coli BL21(DE3)(P9-GFP)(pOL5) cells by rate-zonal and equilibrium flotation centrifugation (42 h). Phi6 represents purified phi6 virions. The molecular weights of selected phi6 structural proteins are indicated on the left (d). e E. coli BL21(DE3)(P9-GFP)(pOL5) cells were visualized using Olympus BX50 microscope and FITC (fluorescein isothiocyanate) filter and visible light. The proteins expressed are indicated on the top, the arrows indicate fluorescent foci inside the cells