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Identification, Characterization and Down-Regulation of Cysteine Protease Genes in Tobacco for Use in Recombinant Protein Production.

Duwadi K, Chen L, Menassa R, Dhaubhadel S - PLoS ONE (2015)

Bottom Line: 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.It was found that the recombinant protein yield in tobacco could be increased by silencing CysP6.Overall results suggest that CysP6 is important in determining the yield of recombinant IL-10 in tobacco leaves.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Western Ontario, London, ON, Canada.

ABSTRACT
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 (CysP) in tobacco. Based on their predicted expression in leaf tissue, 10 candidate CysPs (CysP1-CysP10) were selected for further characterization. The effect of CysP 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 CysP6. Transient expression of CysP6 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 CysP6 is important in determining the yield of recombinant IL-10 in tobacco leaves.

No MeSH data available.


IL-10 accumulation in CysP silenced T1 tobacco lines.Normalized IL-10 levels of different CysP-Si T1 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).
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pone.0130556.g009: IL-10 accumulation in CysP silenced T1 tobacco lines.Normalized IL-10 levels of different CysP-Si T1 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).

Mentions: To study the stability of IL-10 accumulation in CysP silenced tobacco plants, IL-10 levels were measured for a total of 30 different lines in both T0 and T1 generations. The accumulation of IL-10 was stable in most of the lines over successive generations of tobacco (Fig 9). CysP silenced T1 lines, CysP1-Si, CysP2-Si, CysP3-Si and CysP4-Si, all showed lower levels of IL-10 in comparison to their respective IL-10 control plants (represented by same color bars in Fig 9). Lower IL-10 accumulation was also seen for these lines in the T0 generation (Fig 4). Similarly, CysP6 silenced T1 lines showed nearly equal (lines 2 and 15) or higher (lines 8 and 9) levels of IL-10 in comparison to the respective control plant. Both CysP6-Si lines 2 and 15 accumulated higher IL-10 in T0 generation (Fig 9).This suggests that, the effect of CysP silencing on IL-10 accumulation in tobacco remains similar over generations.


Identification, Characterization and Down-Regulation of Cysteine Protease Genes in Tobacco for Use in Recombinant Protein Production.

Duwadi K, Chen L, Menassa R, Dhaubhadel S - PLoS ONE (2015)

IL-10 accumulation in CysP silenced T1 tobacco lines.Normalized IL-10 levels of different CysP-Si T1 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).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4493103&req=5

pone.0130556.g009: IL-10 accumulation in CysP silenced T1 tobacco lines.Normalized IL-10 levels of different CysP-Si T1 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).
Mentions: To study the stability of IL-10 accumulation in CysP silenced tobacco plants, IL-10 levels were measured for a total of 30 different lines in both T0 and T1 generations. The accumulation of IL-10 was stable in most of the lines over successive generations of tobacco (Fig 9). CysP silenced T1 lines, CysP1-Si, CysP2-Si, CysP3-Si and CysP4-Si, all showed lower levels of IL-10 in comparison to their respective IL-10 control plants (represented by same color bars in Fig 9). Lower IL-10 accumulation was also seen for these lines in the T0 generation (Fig 4). Similarly, CysP6 silenced T1 lines showed nearly equal (lines 2 and 15) or higher (lines 8 and 9) levels of IL-10 in comparison to the respective control plant. Both CysP6-Si lines 2 and 15 accumulated higher IL-10 in T0 generation (Fig 9).This suggests that, the effect of CysP silencing on IL-10 accumulation in tobacco remains similar over generations.

Bottom Line: 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.It was found that the recombinant protein yield in tobacco could be increased by silencing CysP6.Overall results suggest that CysP6 is important in determining the yield of recombinant IL-10 in tobacco leaves.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Western Ontario, London, ON, Canada.

ABSTRACT
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 (CysP) in tobacco. Based on their predicted expression in leaf tissue, 10 candidate CysPs (CysP1-CysP10) were selected for further characterization. The effect of CysP 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 CysP6. Transient expression of CysP6 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 CysP6 is important in determining the yield of recombinant IL-10 in tobacco leaves.

No MeSH data available.