9 research outputs found
Tandem fusion of hepatitis B core antigen allows assembly of virus-like particles in bacteria and plants with enhanced capacity to accommodate foreign proteins
The core protein of the hepatitis B virus, HBcAg, assembles into highly immunogenic viruslike particles (HBc VLPs) when expressed in a variety of heterologous systems. Specifically, the major insertion region (MIR) on the HBcAg protein allows the insertion of foreign sequences, which are then exposed on the tips of surface spike structures on the outside of the assembled particle. Here, we present a novel strategy which aids the display of whole proteins on the surface of HBc particles. This strategy, named tandem core, is based on the production of the HBcAg dimer as a single polypeptide chain by tandem fusion of two HBcAg open reading frames. This allows the insertion of large heterologous sequences in only one of the two MIRs in each spike, without compromising VLP formation. We present the use of tandem core technology in both plant and bacterial expression systems. The results show that tandem core particles can be produced with unmodified MIRs, or with one MIR in each tandem dimer modified to contain the entire sequence of GFP or of a camelid nanobody. Both inserted proteins are correctly folded and the nanobody fused to the surface of the tandem core particle (which we name tandibody) retains the ability to bind to its cognate antigen. This technology paves the way for the display of natively folded proteins on the surface of HBc particles either through direct fusion or through non-covalent attachment via a nanobody
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
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 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