Tandem core technology.

<p>a) The structure of a monomeric HBc VLP with one HBcAg dimer shown in a surface representation coloured yellow and green. b) Two HBcAg sequences fused together via a flexible linker makes a tandem core construct, with either full-length (hetero-tandem) or truncated (homo-tandem) C-terminus, and two modifiable major insertion regions (MIRs). c) Structure of a tandem core protein: N-terminal core 1 (in green) is fused via a flexible linker (red) to C-terminal core 2 (yellow). The two views are related by a 90° rotation.</p

Cryo-EM analysis of plant-produced CoHe-GFPL VLPs.

<p>a) Particles were flash-frozen in vitreous ice, then subjected to cryo-electron microscopy. Class averages were obtained from 441 individual particles using EMAN software. The expanded view (lower right) is of an average of all images used. b) 3D reconstruction of the particles using icosahedral symmetry, superimposed on the He map as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120751#pone.0120751.g003" target="_blank">Fig. 3</a>. The CoHe-GFPL map is coloured red-to-blue from the centre of the volume towards its edge; the He map is shown in grey.</p

Cryo-EM of plant-produced τGFP bound with GFP.

<p>a) Class averages computed using Relion of the τGFP particles. b) A 3D reconstruction (resolution estimate 25Å using the “gold standard” cross-FSC at cutoff 0.143) coloured by distance from the centre of the particle (red to blue). The map is shown viewed down a 5-fold axis with the He reconstruction on which the construct was based fitted within (grey surface). The projecting spikes represent density arising from the bound nanobody and GFP but do not occupy every position expected, instead appearing as an average of the density present with the highest intensity at the 2-fold (pseudo- 6-fold) axes and also at the 5-fold axis. These spikes are to some extent artefacts of the icosahedral symmetry imposed on the maps, but are reflected in the spikes also shown in the unaveraged class averages shown in a).</p

τGFP expressed in plants forms VLPs.

<p>a) Predicted structure of the τGFP tandibody protein (Swiss-Prot model): green: core 1, yellow: core 2, pink: anti-GFP nanobody, red: linkers. b) Western blot of crude plant extracts. C: empty vector control, τGFP: tandem HBcAg construct with anti-GFP VHH in the core 2 MIR, μGFP: monomeric HBcAg containing anti-GFP VHH in the MIR. The 39 kDa band found in all plant extracts is non-specific. c) Electron micrograph of plant-produced τGFP particles purified by sucrose cushion. Scale bar 100 nm.</p

Cryo-electron microscopy analysis of E. coli- produced tandem core particles.

<p>a) Surface-rendered views of the reconstructions. Red—hetero-tandem core, contoured at 1σ. Green—homo-tandem core, contoured at 1σ. Blue—difference map, hetero-minus-homo, contoured at 4σ. b) Transverse view across 5-fold axis of the He core with co-ordinates from the HBc crystal structure (Wynne et al., 1999) fitted into the EM density. c) Density profiles of the He (red) and Ho (green) cores generated from translationally-aligned rotational averages. For comparison central sections of the He (upper panel) and Ho (lower panel) maps are shown to the right. A ring of density under the main capsid surface and at a radius of ~90 Å derives from the protamine-like region in He.</p

Tandem cores form VLPs when produced in <i>E</i>. <i>coli</i>.

<p>a) Western blot showing expression in induced (+) and uninduced (-) <i>E. coli</i> of homo- and hetero- tandem core with either 5 (GGS5) or 7 (GGS7) copies of the GGS sequence in the flexible linker between core 1 and core 2. b) Coomassie-stained gel of sucrose gradient fractions of CoHo (<i>E</i>. <i>coli</i> codon-optimised homo-tandem core with GGS7) produced in <i>E</i>. <i>coli</i>. The major band (fraction 2) reacted with anti-HBcAg antibody in western blot analysis. c) Electron micrographs of monomeric (HBcΔ149), codon-optimised homo-tandem (CoHo) and hetero-tandem (CoHe) core particles produced in <i>E</i>. <i>coli</i> and purified by sucrose gradient. Scale bar 100 nm. Arrows indicate smaller (T = 3) particles.</p

Tandem cores can display correctly-folded GFP in plants.

<p>a) White light (top) and UV light (bottom) images of <i>N</i>. <i>benthamiana</i> leaves expressing different constructs via the pEAQ-<i>HT</i> vector. b) UV light image of an ultracentrifuge tube after sucrose gradient purification of plant-produced CoHe-GFPs. The diagram on the right indicates the location of the sucrose layers and their concentration. The area represented in green is the clarified plant lysate. c) Electron micrograph of plant-produced CoHe-GFPs VLPs purified by sucrose gradient. Scale bar 100 nm.</p