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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 candidate CysP silenced T0 tobacco lines.IL-10 accumulation remains lower in comparison to the controls in T0 generation of CysP1-CysP10 (except CysP6) 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.
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pone.0130556.g004: IL-10 accumulation in candidate CysP silenced T0 tobacco lines.IL-10 accumulation remains lower in comparison to the controls in T0 generation of CysP1-CysP10 (except CysP6) 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.

Mentions: To determine the effect of CysP silencing in the accumulation of recombinant IL-10, we measured IL-10 levels in multiple independent transgenic silencing lines for CysP1-CysP4, CysP6-CysP10 and CysPAll. Except for several CysP6Si lines and CysP8Si-3, there was no increase in IL-10 accumulation compared to the IL-10 control plants in any other lines (Fig 4). Since these CysP silenced lines were generated over different periods of time, IL-10 control plants were also grown at the same time for an appropriate comparison of IL-10 levels. It was found that the accumulation of IL-10 varied among the controls, even though the plants were grown under similar conditions. Thus, IL-10 accumulations for different T0 lines are shown in normalized fold levels.


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 candidate CysP silenced T0 tobacco lines.IL-10 accumulation remains lower in comparison to the controls in T0 generation of CysP1-CysP10 (except CysP6) 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130556.g004: IL-10 accumulation in candidate CysP silenced T0 tobacco lines.IL-10 accumulation remains lower in comparison to the controls in T0 generation of CysP1-CysP10 (except CysP6) 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.
Mentions: To determine the effect of CysP silencing in the accumulation of recombinant IL-10, we measured IL-10 levels in multiple independent transgenic silencing lines for CysP1-CysP4, CysP6-CysP10 and CysPAll. Except for several CysP6Si lines and CysP8Si-3, there was no increase in IL-10 accumulation compared to the IL-10 control plants in any other lines (Fig 4). Since these CysP silenced lines were generated over different periods of time, IL-10 control plants were also grown at the same time for an appropriate comparison of IL-10 levels. It was found that the accumulation of IL-10 varied among the controls, even though the plants were grown under similar conditions. Thus, IL-10 accumulations for different T0 lines are shown in normalized fold levels.

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.