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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.


CysP6 localizes to the ER.Agrobacterium the carrying plasmids pCysP6-YFP and pER-CFP (ER control) were infiltrated in Nicotiana benthamiana (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.
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pone.0130556.g007: CysP6 localizes to the ER.Agrobacterium the carrying plasmids pCysP6-YFP and pER-CFP (ER control) were infiltrated in Nicotiana benthamiana (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.

Mentions: To evaluate the possibility that IL-10 is degraded by CysP6 in the secretory pathway, the subcellular localization of CysP6 was determined. A translational fusion of CysP6 and YFP was created (Fig 7A), and transiently co-expressed with ER-targeted CFP (control) in N. benthamiana leaf epidermal cells, followed by confocal microscopy. A typical ER-pattern showing net-like structures and co-localization of CysP6-YFP protein with ER-targeted CFP was observed, indicating that CysP6 localizes in the ER in tobacco leaf cells (Fig 7B).


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)

CysP6 localizes to the ER.Agrobacterium the carrying plasmids pCysP6-YFP and pER-CFP (ER control) were infiltrated in Nicotiana benthamiana (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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130556.g007: CysP6 localizes to the ER.Agrobacterium the carrying plasmids pCysP6-YFP and pER-CFP (ER control) were infiltrated in Nicotiana benthamiana (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.
Mentions: To evaluate the possibility that IL-10 is degraded by CysP6 in the secretory pathway, the subcellular localization of CysP6 was determined. A translational fusion of CysP6 and YFP was created (Fig 7A), and transiently co-expressed with ER-targeted CFP (control) in N. benthamiana leaf epidermal cells, followed by confocal microscopy. A typical ER-pattern showing net-like structures and co-localization of CysP6-YFP protein with ER-targeted CFP was observed, indicating that CysP6 localizes in the ER in tobacco leaf cells (Fig 7B).

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.