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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.


Related in: MedlinePlus

RNAi silencing in tobacco.RNAi constructs were generated for all candidate CysPs and the presence of CysP double inserts in the silencing vectors were confirmed using primer combinations as shown by the arrowheads (A). Stable CysP silenced tobacco lines were generated using the RNAi constructs (B). G7 tobacco lines overexpressing IL-10 protein were grown in magenta boxes. Leaf discs were cut and co-cultured with the Agrobacterium cultures containing the RNAi construct. Calli were regenerated from transformed cells and subcultured in differentiation medium containing BASTA. Independent CysP silenced transgenic lines were obtained from the individual callus and transferred to the rooting media before transferring them to the greenhouse.
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pone.0130556.g003: RNAi silencing in tobacco.RNAi constructs were generated for all candidate CysPs and the presence of CysP double inserts in the silencing vectors were confirmed using primer combinations as shown by the arrowheads (A). Stable CysP silenced tobacco lines were generated using the RNAi constructs (B). G7 tobacco lines overexpressing IL-10 protein were grown in magenta boxes. Leaf discs were cut and co-cultured with the Agrobacterium cultures containing the RNAi construct. Calli were regenerated from transformed cells and subcultured in differentiation medium containing BASTA. Independent CysP silenced transgenic lines were obtained from the individual callus and transferred to the rooting media before transferring them to the greenhouse.

Mentions: To identify CysPs that influence IL-10 accumulation in tobacco, RNAi silencing of selected CysPs was performed. Unique regions were identified for each of the CysP sequences to facilitate targeted gene silencing. RNAi vectors with the specific CysP inserts were screened using two different primer combinations by PCR, which produced amplicons of different sizes. The first amplicon contained the larger intron (In-1) and the second amplicon contained the smaller intron (In-2) region (Fig 3A). A low alkaloid tobacco G7 line constitutively overexpressing IL-10 [32] was used for transforming the silencing constructs. Fig 3B outlines the process of generating CysP silenced tobacco plants and the approximate time required to proceed from one stage to the other. In approximately 16 weeks from initial transformation, fully grown plants were obtained. A total of 82 independent transgenic lines were generated, and utilized for the evaluation of CysP transcript and IL-10 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)

RNAi silencing in tobacco.RNAi constructs were generated for all candidate CysPs and the presence of CysP double inserts in the silencing vectors were confirmed using primer combinations as shown by the arrowheads (A). Stable CysP silenced tobacco lines were generated using the RNAi constructs (B). G7 tobacco lines overexpressing IL-10 protein were grown in magenta boxes. Leaf discs were cut and co-cultured with the Agrobacterium cultures containing the RNAi construct. Calli were regenerated from transformed cells and subcultured in differentiation medium containing BASTA. Independent CysP silenced transgenic lines were obtained from the individual callus and transferred to the rooting media before transferring them to the greenhouse.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130556.g003: RNAi silencing in tobacco.RNAi constructs were generated for all candidate CysPs and the presence of CysP double inserts in the silencing vectors were confirmed using primer combinations as shown by the arrowheads (A). Stable CysP silenced tobacco lines were generated using the RNAi constructs (B). G7 tobacco lines overexpressing IL-10 protein were grown in magenta boxes. Leaf discs were cut and co-cultured with the Agrobacterium cultures containing the RNAi construct. Calli were regenerated from transformed cells and subcultured in differentiation medium containing BASTA. Independent CysP silenced transgenic lines were obtained from the individual callus and transferred to the rooting media before transferring them to the greenhouse.
Mentions: To identify CysPs that influence IL-10 accumulation in tobacco, RNAi silencing of selected CysPs was performed. Unique regions were identified for each of the CysP sequences to facilitate targeted gene silencing. RNAi vectors with the specific CysP inserts were screened using two different primer combinations by PCR, which produced amplicons of different sizes. The first amplicon contained the larger intron (In-1) and the second amplicon contained the smaller intron (In-2) region (Fig 3A). A low alkaloid tobacco G7 line constitutively overexpressing IL-10 [32] was used for transforming the silencing constructs. Fig 3B outlines the process of generating CysP silenced tobacco plants and the approximate time required to proceed from one stage to the other. In approximately 16 weeks from initial transformation, fully grown plants were obtained. A total of 82 independent transgenic lines were generated, and utilized for the evaluation of CysP transcript and IL-10 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.


Related in: MedlinePlus