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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 tobacco plants of different stages.IL-10 accumulation in 4 weeks and 7 weeks old CysP6 silenced tobacco lines. IL-10 accumulation increased consistently in CysP6 silenced lines and IL-10 controls with the age. The numbers indicate different independent transgenic lines carrying CysP6 silencing construct. TSP, total soluble protein; Si, silenced.
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pone.0130556.g008: IL-10 accumulation in tobacco plants of different stages.IL-10 accumulation in 4 weeks and 7 weeks old CysP6 silenced tobacco lines. IL-10 accumulation increased consistently in CysP6 silenced lines and IL-10 controls with the age. The numbers indicate different independent transgenic lines carrying CysP6 silencing construct. TSP, total soluble protein; Si, silenced.

Mentions: To evaluate if the age of transgenic tobacco plants affects the production of recombinant protein, IL-10 levels in CysP6 silenced lines and IL-10 controls were compared at two different time points. The plants were grown simultaneously in the greenhouse and IL-10 level was measured when they were either 4- or 7-weeks old (time after the transfer of plants from rooting media to soil). The plants were approximately 0.5 ft. tall at 4 weeks of age and approximately 1.5 ft. tall at 7 weeks of age. As shown in Fig 8, the levels of IL-10 accumulation in the leaf tissue increased as plants progressed to maturity in both CysP6 silenced and IL-10 control plants. Moreover, neither abnormal growth nor any morphological differences were observed for the CysP silenced lines compared to IL-10 controls.


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 tobacco plants of different stages.IL-10 accumulation in 4 weeks and 7 weeks old CysP6 silenced tobacco lines. IL-10 accumulation increased consistently in CysP6 silenced lines and IL-10 controls with the age. The numbers indicate different independent transgenic lines carrying CysP6 silencing construct. TSP, total soluble protein; Si, silenced.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130556.g008: IL-10 accumulation in tobacco plants of different stages.IL-10 accumulation in 4 weeks and 7 weeks old CysP6 silenced tobacco lines. IL-10 accumulation increased consistently in CysP6 silenced lines and IL-10 controls with the age. The numbers indicate different independent transgenic lines carrying CysP6 silencing construct. TSP, total soluble protein; Si, silenced.
Mentions: To evaluate if the age of transgenic tobacco plants affects the production of recombinant protein, IL-10 levels in CysP6 silenced lines and IL-10 controls were compared at two different time points. The plants were grown simultaneously in the greenhouse and IL-10 level was measured when they were either 4- or 7-weeks old (time after the transfer of plants from rooting media to soil). The plants were approximately 0.5 ft. tall at 4 weeks of age and approximately 1.5 ft. tall at 7 weeks of age. As shown in Fig 8, the levels of IL-10 accumulation in the leaf tissue increased as plants progressed to maturity in both CysP6 silenced and IL-10 control plants. Moreover, neither abnormal growth nor any morphological differences were observed for the CysP silenced lines compared to IL-10 controls.

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.