Schematic representation of different domains in papain and otubain family CysPs.

<p>Candidate CysPs having specific domain types are indicated. The diagram is not drawn to scale. I29, Cathepsin propeptide inhibitor domain; Peptidase_C1, Papain family domain; GRN, Granulin like repeats; Peptidase_C65, Otubain family domain.</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 candidate <i>CysP</i> silenced T₀ tobacco lines.

<p>IL-10 accumulation remains lower in comparison to the controls in T_<b>0</b> generation of <i>CysP1</i>-<i>CysP10</i> (except <i>CysP6</i>) silenced lines. Y-axis represents the normalized value of IL-10/TSP for different independent transgenic lines grown at different time period. Blue, red, black and green bars are normalized against their respective color coded IL-10 level in IL-10 control plant. Numbers above the bar represent actual IL-10 level in ng/mg of total soluble protein in control plants. Si, silenced.</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 <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

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

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

Multiple sequence alignment of Peptidase_C1 (Papain) family candidate CysPs.

<p>Predicted amino acid sequences of a papain, NTCP23 (CysP1), CysP2, CysP4, CysP5, NTCP6 (CysP6), CysP7, CysP8 and CysP10 were aligned using CLUSTALW and shaded using BOXSHADE 3.21. Identical and conserved amino acids are shaded by dark and light grey, respectively. (*) indicates catalytic residues cysteine (C), histidine (H) and asparagine (N). (*) indicates the disulfide bridge forming cysteine residues. Red box indicates endoplasmic reticulum retention signal KDEL in CysP2 and CysP10. The conserved ERFNIN motif in the propeptide region is double-underlined. C-terminal amino acids of CysP6 and CysP8 beyond the positions 410 and 362, respectively, are not shown in the alignment.</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