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

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

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

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