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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 CysP6 silenced T0 tobacco lines.Measurement of IL-10 level in independent T0CysP6 silenced lines by dsELISA. Data label above the bar represents IL-10 level of individual CysP6 silenced lines expressed as ng/mg of total soluble protein. + indicates the CysP6 silenced lines with higher level of IL-10 accumulation in comparison to the IL-10 control. TSP, total soluble protein; Si, silenced
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pone.0130556.g005: IL-10 accumulation in CysP6 silenced T0 tobacco lines.Measurement of IL-10 level in independent T0CysP6 silenced lines by dsELISA. Data label above the bar represents IL-10 level of individual CysP6 silenced lines expressed as ng/mg of total soluble protein. + indicates the CysP6 silenced lines with higher level of IL-10 accumulation in comparison to the IL-10 control. TSP, total soluble protein; Si, silenced

Mentions: A closer look at CysP6Si lines revealed that IL-10 levels were higher in 5 out of 13 CysP6 silenced lines compared to the control (Fig 5). IL-10 level was increased by 1.6- fold in CysP6Si-15 as compared to the control. Three lines, CysP6Si-4, CysP6Si-7 and CysP6Si-13, displayed similar level of IL-10 accumulation in comparison to the control. The results indicated that silencing of CysP6 positively affects IL-10 accumulation in T0 transgenic tobacco lines.


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 CysP6 silenced T0 tobacco lines.Measurement of IL-10 level in independent T0CysP6 silenced lines by dsELISA. Data label above the bar represents IL-10 level of individual CysP6 silenced lines expressed as ng/mg of total soluble protein. + indicates the CysP6 silenced lines with higher level of IL-10 accumulation in comparison to the IL-10 control. TSP, total soluble protein; Si, silenced
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130556.g005: IL-10 accumulation in CysP6 silenced T0 tobacco lines.Measurement of IL-10 level in independent T0CysP6 silenced lines by dsELISA. Data label above the bar represents IL-10 level of individual CysP6 silenced lines expressed as ng/mg of total soluble protein. + indicates the CysP6 silenced lines with higher level of IL-10 accumulation in comparison to the IL-10 control. TSP, total soluble protein; Si, silenced
Mentions: A closer look at CysP6Si lines revealed that IL-10 levels were higher in 5 out of 13 CysP6 silenced lines compared to the control (Fig 5). IL-10 level was increased by 1.6- fold in CysP6Si-15 as compared to the control. Three lines, CysP6Si-4, CysP6Si-7 and CysP6Si-13, displayed similar level of IL-10 accumulation in comparison to the control. The results indicated that silencing of CysP6 positively affects IL-10 accumulation in T0 transgenic tobacco lines.

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.