Comparing the effects of expression vector and subcellular localization on the expression of LALF_32-51-E7.

<p><b>(A),</b> Leaf physiology monitored over time after vacuum infiltration with pRIC3.0-LFLF_32-51-E7, pRIC3.0-cTP-LFLF_32-51-E7 and pRIC3.0 empty. All cultures used were set to an OD₆₀₀ of 1.0. Shown are representative leaves on 3, 5 and 7 dpi of three independent repeats. <b>(B)</b> The effect of expression vector on LALF_32-51-E7 accumulation. Left panel, equal volume western blots of LALF_32-51-E7 expression from pTRAc using an antibody dilution of 1:1,000. (+), purified <i>E</i>. <i>coli</i>-derived LALF_32-51-E7. (-), pRIC3.0 empty vector crude extract. Black arrow indicates the expected position of LALF_32-51-E7, ≈22 kDa. Blue arrows show higher molecular weight aggregates. Right panel, analysis of 50 μg of TSP of pRIC3.0-LALF_32-51-E7 and pRIC3.0-cTP-LALF_32-51-E7 crude extracts. Western blot using an antibody dilution of 1:5,000 (top) and corresponding AquaStained SDS-PAGE gels showing a native protein of approximately 50 kDa as an internal control for equal TSP loading (bottom). (-), pRIC3.0 empty vector crude extract. (+), <i>E</i>. <i>coli</i>-derived LALF_32-51-E7 inclusion bodies. Arrow indicates the position of LALF_32-51-E7, ≈22 kDa. Mw, molecular weight marker. TSP, total soluble proteins. <b>(C)</b> The effect of subcellular localization on LALF_32-51-E7 accumulation determined by the %TSP.</p

The effects of silencing suppressors on the expression of LALF_32-51-E7.

<p><b>(A)</b> Leaf physiology monitored over 7 days after vacuum co-infiltration of silencing suppressors and pRIC3.0-LALF _32-51-E7 or pRIC3.0-cTP-LALF _32-51-E7. All cultures used were set to an OD₆₀₀ of 1.0. Shown are representative leaves on 3, 5 and 7 dpi. pRIC3.0 empty vector was used as a negative control. <b>(B)</b> Analysis of 50 μg of TSP crude extracts of leaves vacuum-infiltrated with or without a silencing suppressor on the best expression dpi. Top panel, western blot using an antibody dilution of 1:5,000. Bottom panel, corresponding AquaStained SDS-PAGE gel showing a native protein of approximately 50 kDa as an internal control for equal TSP loading. (+), <i>E</i>. <i>coli</i>-derived LALF _32-51-E7 inclusion bodies. Arrow indicates the position of LALF _32-51-E7 ≈22 kDa. Shown here are representative images of two independent repeats. Mw, molecular weight marker. TSP, total soluble protein.</p

Purification of LALF_32-51-E7 using Ni²⁺ column chromatography.

<p><b>(A)</b>, LALF_32-51-E7 purification chromatogram. Blue curve, A₂₈₀ representing protein levels. Light-green curve, elution buffer gradient. Arrow shows the elution peak corresponding to elution fractions 1–7. Black box, elution fractions 5–7 corresponding to LALF_32-51-E7. <b>(B)</b>, analysis of relevant fractions by AquaStained SDS-PAGE gel (left panel) and anti-E7 polyclonal antibody western blot using dilution of 1:5,000 (right panel). (+), <i>E</i>. <i>coli</i>-LALF_32-51-E7 inclusion bodies. CE, LALF_32-51-E7 crude extract from 50 g fresh leaf weight. FT, flow through. E4-E13, elution fractions 4–13. Arrows point at the doublet bands representing LALF_32-51-E7 ≈ 20 and 22 kDa. Mw, molecular weight marker.</p

Comparing the effects of expression vector and subcellular localization on the expression of LALF_32-51-E7.

<p><b>(A),</b> Leaf physiology monitored over time after vacuum infiltration with pRIC3.0-LFLF_32-51-E7, pRIC3.0-cTP-LFLF_32-51-E7 and pRIC3.0 empty. All cultures used were set to an OD₆₀₀ of 1.0. Shown are representative leaves on 3, 5 and 7 dpi of three independent repeats. <b>(B)</b> The effect of expression vector on LALF_32-51-E7 accumulation. Left panel, equal volume western blots of LALF_32-51-E7 expression from pTRAc using an antibody dilution of 1:1,000. (+), purified <i>E</i>. <i>coli</i>-derived LALF_32-51-E7. (-), pRIC3.0 empty vector crude extract. Black arrow indicates the expected position of LALF_32-51-E7, ≈22 kDa. Blue arrows show higher molecular weight aggregates. Right panel, analysis of 50 μg of TSP of pRIC3.0-LALF_32-51-E7 and pRIC3.0-cTP-LALF_32-51-E7 crude extracts. Western blot using an antibody dilution of 1:5,000 (top) and corresponding AquaStained SDS-PAGE gels showing a native protein of approximately 50 kDa as an internal control for equal TSP loading (bottom). (-), pRIC3.0 empty vector crude extract. (+), <i>E</i>. <i>coli</i>-derived LALF_32-51-E7 inclusion bodies. Arrow indicates the position of LALF_32-51-E7, ≈22 kDa. Mw, molecular weight marker. TSP, total soluble proteins. <b>(C)</b> The effect of subcellular localization on LALF_32-51-E7 accumulation determined by the %TSP.</p

Enhanced LALF_32-51-E7 extraction strategy.

<p>Leaf material expressing LALF_32-51-E7 was ground-up with liquid nitrogen and washed 3 times with PBS (W1-3). LALF_32-51-E7 was solubilized from the leaf material with extraction buffer containing 6 M or 8 M urea (6M and 8M, respectively). The final extracts are compared to a crude extract sample prepared using the previous extraction strategy (PE). (+), purified <i>E</i>. <i>coli</i>-produced LALF_32-51-E7. Left panel, Coomassie-stained SDS-PAGE gel. Right panel, western blot using an antibody dilution 1:5,000. Arrows indicate the position of LALF_32-51-E7 ≈ 22 kDa. Mw, molecular weight marker.</p

Construct generation for the expression of LALF_32-51-E7 in <i>N</i>. <i>benthamiana</i>.

<p>The LALF_32-51-E7 sequence was plant codon-optimized and inserted into the self-replicating plant expression vector pRIC3.0, with and without a chloroplast targeting peptide (cTP) signal. Light-blue arrows, restriction enzyme sites. CAMV-promoter, the cauliflower mosaic virus 35S constitutive promoter. 6xHIS, hexa-histidine tag. Not drawn to scale.</p

Expression optimization of a cell membrane-penetrating human papillomavirus type 16 therapeutic vaccine candidate in <i>Nicotiana benthamiana</i>

<div><p>High-risk human papillomaviruses (hr-HPVs) cause cervical cancer, the fourth most common cancer in women worldwide. A HPV-16 candidate therapeutic vaccine, LALF_32-51-E7, was developed by fusing a modified E7 protein to a bacterial cell-penetrating peptide (LALF): this elicited both tumour protection and regression in pre-clinical immunization studies. In the current study, we investigated the potential for producing LALF_32-51-E7 in a plant expression system by evaluating the effect of subcellular localization and usage of different expression vectors and gene silencing suppressors. The highest expression levels of LALF_32-51-E7 were obtained by using a self-replicating plant expression vector and chloroplast targeting, which increased its accumulation by 27-fold compared to cytoplasmic localization. The production and extraction of LALF_32-51-E7 was scaled-up and purification optimized by affinity chromatography. If further developed, this platform could potentially allow for the production of a more affordable therapeutic vaccine for HPV-16. This would be extremely relevant in the context of developing countries, where cervical cancer and other HPV-related malignancies are most prevalent, and where the population have limited or no access to preventative vaccines due to their typical high costs.</p></div

Gus assays to test for cryptic splice sites in the MSV long intergenic region.

<p>A) Gus expression cassettes used in the assays. B) Expression of Gus from p35S-GSLIR²⁴¹ (test construct) as a ratio to p35S-GS (positive control construct), four days after bombardment. Each bar is an average of three replicates; error bars represent 95% confidence intervals. Negative = negative control (protein extract from a non-bombarded Black Mexican sweet sample).</p

Schematic diagram of synthesised constructs, with restriction enzyme sites incorporated for subsequent cloning.

<p>A) pSPLIT<i>rep</i>^1-219Rb-35S containing “modules” that could be removed and replaced with other sequences by restriction digest. B) Illustration showing how the <i>rep</i>^1-219Rb- transgene was split at the first AGGC (nucleotides 155, 156, 157 and 158 with respect to the start codon). The exon 2, cloned at the 5′ terminus of the split gene cassette in A) therefore began with GC, and the exon 1, cloned at the 3′ terminus, ended in AG. C) The synthesised <i>rep</i>^III-Rb- (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105932#pone-0105932-g001" target="_blank">Fig. 1A</a> for the full-length gene product) exon 2, preceded by the 3′-terminal half of the syntron, flanked by <i>Swa</i>I and <i>Spe</i>I RE sites. The 3′-terminal syntron/<i>rep</i>^1-219Rb- exon 2 in pSPLIT<i>rep</i>^1-219Rb-35S was replaced by the 3′-terminal syntron/<i>rep</i>^III-Rb- exon 2 to create pSPLIT<i>rep</i>^III-Rb-35S. Exon 1 remained the same for both constructs since they share the same 5′-terminal 156 bp. Similarly, other modules were exchanged to create further constructs, such as the CaMV 35S promoter for the maize ubiquitin promoter etc (see text for details).</p

Primer sequences.

1<p>Underlined letters highlight engineered restriction enzyme (RE) sites (names of the introduced RE sites are incorporated in the primer names).</p><p>Primer sequences.</p