IL-10 accumulation in <i>CysP6</i> silenced T₀ tobacco lines.

<p>Measurement of IL-10 level in independent T_<b>0</b><i>CysP6</i> silenced lines by dsELISA. Data label above the bar represents IL-10 level of individual <i>CysP6</i> silenced lines expressed as ng/mg of total soluble protein. <b>+</b> indicates the <i>CysP6</i> silenced lines with higher level of IL-10 accumulation in comparison to the IL-10 control. TSP, total soluble protein; Si, silenced</p

Detail domain information of candidate tobacco CysPs.

<p>Numbers in the domains represent amino acids.</p><p>*complete coding region sequence was not available.</p><p>X, absent in respective CysPs.</p><p>Detail domain information of candidate tobacco CysPs.</p

Identification, Characterization and Down-Regulation of <i>Cysteine Protease</i> Genes in Tobacco for Use in Recombinant Protein Production

<div><p>Plants are an attractive host system for pharmaceutical protein production. Many therapeutic proteins have been produced and scaled up in plants at a low cost compared to the conventional microbial and animal-based systems. The main technical challenge during this process is to produce sufficient levels of recombinant proteins in plants. Low yield is generally caused by proteolytic degradation during expression and downstream processing of recombinant proteins. The yield of human therapeutic interleukin (IL)-10 produced in transgenic tobacco leaves was found to be below the critical level, and may be due to degradation by tobacco proteases. Here, we identified a total of 60 putative cysteine protease genes (<i>CysP</i>) in tobacco. Based on their predicted expression in leaf tissue, 10 candidate <i>CysP</i>s (<i>CysP1</i>-<i>CysP10</i>) were selected for further characterization. The effect of <i>CysP</i> gene silencing on IL-10 accumulation was examined in tobacco. It was found that the recombinant protein yield in tobacco could be increased by silencing <i>CysP6</i>. Transient expression of <i>CysP6</i> silencing construct also showed an increase in IL-10 accumulation in comparison to the control. Moreover, CysP6 localizes to the endoplasmic reticulum (ER), suggesting that ER may be the site of IL-10 degradation. Overall results suggest that <i>CysP6</i> is important in determining the yield of recombinant IL-10 in tobacco leaves.</p></div

IL-10 accumulation in tobacco plants of different stages.

<p>IL-10 accumulation in 4 weeks and 7 weeks old <i>CysP6</i> silenced tobacco lines. IL-10 accumulation increased consistently in <i>CysP6</i> silenced lines and IL-10 controls with the age. The numbers indicate different independent transgenic lines carrying <i>CysP6</i> silencing construct. TSP, total soluble protein; Si, silenced.</p

RNAi silencing in tobacco.

<p>RNAi constructs were generated for all candidate <i>CysP</i>s and the presence of <i>CysP</i> double inserts in the silencing vectors were confirmed using primer combinations as shown by the arrowheads (A). Stable <i>CysP</i> silenced tobacco lines were generated using the RNAi constructs (B). G7 tobacco lines overexpressing IL-10 protein were grown in magenta boxes. Leaf discs were cut and co-cultured with the <i>Agrobacterium</i> cultures containing the RNAi construct. Calli were regenerated from transformed cells and subcultured in differentiation medium containing BASTA. Independent <i>CysP</i> silenced transgenic lines were obtained from the individual callus and transferred to the rooting media before transferring them to the greenhouse.</p

Comparison of published tobacco <i>CysP</i> sequences with tentative contig sequences from tobacco EST database.

<p>^aClosed tentative contig in bold letters (candidate CysPs in parenthesis) share a high degree of amino acid identity with the respective published CysP sequences</p><p>Comparison of published tobacco <i>CysP</i> sequences with tentative contig sequences from tobacco EST database.</p

Silencing of <i>CysP6</i> in tobacco using transient assay and stable transgenic lines.

<p>Leaves from seven weeks old G7 tobacco plants (4^th, 5^th and 6^th from the top) were infiltrated with the <i>CysP6</i> silencing (CysP6-Si) and empty vector constructs (Si vec.), and the tissues were used to determine the <i>CysP6</i> transcript and IL-10 accumulation levels. <i>CysP6</i> expressions were checked in CysP6-Si and Si Vec. infiltrated tissues, 4 days post infiltration. The numbers 1–7 indicate the biological replicates (B). The accumulation of IL-10 was measured in all the <i>CysP6</i> silencing and vector only infiltrated tissues. The IL-10/TSP values are average of 7 biological replicates and asterisk (*) represents the significant difference in IL-10 level between CysP6-Si and vector only control using student <i>t</i>-test at 95% confidence level. Si, Silenced; Vec., Vector (C). Comparison of <i>CysP6</i> expression and IL-10 accumulation in CysP6Si T_<b>1</b> lines. Blue bars represent IL-10/TSP normalized to IL-10 control and orange bars represent fold expression of <i>CysP6</i> normalized to <i>Actin</i>. Three biological and three technical replicates were used. Error bars represent the standard errors of the biological and technical replicates (D). Asterisks (*) indicates the significant difference in <i>CysP6</i> expression as compared to control, and hashtags (#) indicates the significant difference in IL-10 levels between CysP6-Si lines and IL-10 controls using Mann-Whitney <i>U</i> test (for qPCR data) and students <i>t</i>-test (for ELISA data) at P < 0.05.</p

IL-10 accumulation in <i>CysP</i> silenced T₁ tobacco lines.

<p>Normalized IL-10 levels of different CysP-Si T_<b>1</b> lines and controls. Green, blue and red bars are normalized to the IL-10 levels in their respective control plants (IL-10 control, on the right). Numbers above the bar represent the actual IL-10 levels in ng/mg of total soluble proteins. Error bars represent the mean of at least two biological replicates for all the lines except for CysP6-Si lines 8 and 15 (3 biological replicates).</p

List of putative <i>CysPs</i>, tissue expression and predicted protein size of candidate CysPs selected for silencing in tobacco.

<p>TC, tentative contig sequence; AA, amino acid; L, leaf; F, flower; R, root; W, whole plant, BY-2, <i>Nicotiana tabacum cv</i>. Bright Yellow cell line;-, partial <i>CysP</i> sequences</p><p>List of putative <i>CysPs</i>, tissue expression and predicted protein size of candidate CysPs selected for silencing in tobacco.</p

CysP6 localizes to the ER.

<p><i>Agrobacterium</i> the carrying plasmids pCysP6-YFP and pER-CFP (ER control) were infiltrated in <i>Nicotiana benthamiana</i> (A). The localization was visualized after 48 hours using confocal microscopy (B). CysP6-YFP was visualized in the networks of the ER and co-localizes with the control, ER-CFP. Scale bars, 15 μm.</p