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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.


Silencing of CysP6 in tobacco using transient assay and stable transgenic lines.Leaves from seven weeks old G7 tobacco plants (4th, 5th and 6th from the top) were infiltrated with the CysP6 silencing (CysP6-Si) and empty vector constructs (Si vec.), and the tissues were used to determine the CysP6 transcript and IL-10 accumulation levels. CysP6 expressions were checked in CysP6-Si and Si Vec. infiltrated tissues, 4 days post infiltration. The numbers 1–7 indicate the biological replicates (B). The accumulation of IL-10 was measured in all the CysP6 silencing and vector only infiltrated tissues. The IL-10/TSP values are average of 7 biological replicates and asterisk (*) represents the significant difference in IL-10 level between CysP6-Si and vector only control using student t-test at 95% confidence level. Si, Silenced; Vec., Vector (C). Comparison of CysP6 expression and IL-10 accumulation in CysP6Si T1 lines. Blue bars represent IL-10/TSP normalized to IL-10 control and orange bars represent fold expression of CysP6 normalized to Actin. Three biological and three technical replicates were used. Error bars represent the standard errors of the biological and technical replicates (D). Asterisks (*) indicates the significant difference in CysP6 expression as compared to control, and hashtags (#) indicates the significant difference in IL-10 levels between CysP6-Si lines and IL-10 controls using Mann-Whitney U test (for qPCR data) and students t-test (for ELISA data) at P < 0.05.
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pone.0130556.g006: Silencing of CysP6 in tobacco using transient assay and stable transgenic lines.Leaves from seven weeks old G7 tobacco plants (4th, 5th and 6th from the top) were infiltrated with the CysP6 silencing (CysP6-Si) and empty vector constructs (Si vec.), and the tissues were used to determine the CysP6 transcript and IL-10 accumulation levels. CysP6 expressions were checked in CysP6-Si and Si Vec. infiltrated tissues, 4 days post infiltration. The numbers 1–7 indicate the biological replicates (B). The accumulation of IL-10 was measured in all the CysP6 silencing and vector only infiltrated tissues. The IL-10/TSP values are average of 7 biological replicates and asterisk (*) represents the significant difference in IL-10 level between CysP6-Si and vector only control using student t-test at 95% confidence level. Si, Silenced; Vec., Vector (C). Comparison of CysP6 expression and IL-10 accumulation in CysP6Si T1 lines. Blue bars represent IL-10/TSP normalized to IL-10 control and orange bars represent fold expression of CysP6 normalized to Actin. Three biological and three technical replicates were used. Error bars represent the standard errors of the biological and technical replicates (D). Asterisks (*) indicates the significant difference in CysP6 expression as compared to control, and hashtags (#) indicates the significant difference in IL-10 levels between CysP6-Si lines and IL-10 controls using Mann-Whitney U test (for qPCR data) and students t-test (for ELISA data) at P < 0.05.

Mentions: To further examine the effect of CysP6 silencing on IL-10 accumulation levels, transient silencing experiments were performed in tobacco plants that were 1.5 ft tall. A. tumefaciens containing CysP6-Si construct was infiltrated into tobacco leaf epidermal cells and samples were collected for evaluating CysP6 transcript and IL-10 protein accumulation. Our RT-PCR analysis using gene specific primers did not detect any CysP6 transcript in all 7 biological replicates whereas it was present in vector-only plants (Fig 6A). This suggests that CysP6 was efficiently silenced by transient expression of the silencing construct. Furthermore, IL-10 levels were significantly higher in all 7 biological replicates which were infiltrated with the CysP6 silencing construct, than tissues infiltrated with the vector-only constructs (Fig 6B). These results demonstrated that transient silencing CysP6 results in the increase in IL-10 accumulation in G7 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)

Silencing of CysP6 in tobacco using transient assay and stable transgenic lines.Leaves from seven weeks old G7 tobacco plants (4th, 5th and 6th from the top) were infiltrated with the CysP6 silencing (CysP6-Si) and empty vector constructs (Si vec.), and the tissues were used to determine the CysP6 transcript and IL-10 accumulation levels. CysP6 expressions were checked in CysP6-Si and Si Vec. infiltrated tissues, 4 days post infiltration. The numbers 1–7 indicate the biological replicates (B). The accumulation of IL-10 was measured in all the CysP6 silencing and vector only infiltrated tissues. The IL-10/TSP values are average of 7 biological replicates and asterisk (*) represents the significant difference in IL-10 level between CysP6-Si and vector only control using student t-test at 95% confidence level. Si, Silenced; Vec., Vector (C). Comparison of CysP6 expression and IL-10 accumulation in CysP6Si T1 lines. Blue bars represent IL-10/TSP normalized to IL-10 control and orange bars represent fold expression of CysP6 normalized to Actin. Three biological and three technical replicates were used. Error bars represent the standard errors of the biological and technical replicates (D). Asterisks (*) indicates the significant difference in CysP6 expression as compared to control, and hashtags (#) indicates the significant difference in IL-10 levels between CysP6-Si lines and IL-10 controls using Mann-Whitney U test (for qPCR data) and students t-test (for ELISA data) at P < 0.05.
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Related In: Results  -  Collection

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pone.0130556.g006: Silencing of CysP6 in tobacco using transient assay and stable transgenic lines.Leaves from seven weeks old G7 tobacco plants (4th, 5th and 6th from the top) were infiltrated with the CysP6 silencing (CysP6-Si) and empty vector constructs (Si vec.), and the tissues were used to determine the CysP6 transcript and IL-10 accumulation levels. CysP6 expressions were checked in CysP6-Si and Si Vec. infiltrated tissues, 4 days post infiltration. The numbers 1–7 indicate the biological replicates (B). The accumulation of IL-10 was measured in all the CysP6 silencing and vector only infiltrated tissues. The IL-10/TSP values are average of 7 biological replicates and asterisk (*) represents the significant difference in IL-10 level between CysP6-Si and vector only control using student t-test at 95% confidence level. Si, Silenced; Vec., Vector (C). Comparison of CysP6 expression and IL-10 accumulation in CysP6Si T1 lines. Blue bars represent IL-10/TSP normalized to IL-10 control and orange bars represent fold expression of CysP6 normalized to Actin. Three biological and three technical replicates were used. Error bars represent the standard errors of the biological and technical replicates (D). Asterisks (*) indicates the significant difference in CysP6 expression as compared to control, and hashtags (#) indicates the significant difference in IL-10 levels between CysP6-Si lines and IL-10 controls using Mann-Whitney U test (for qPCR data) and students t-test (for ELISA data) at P < 0.05.
Mentions: To further examine the effect of CysP6 silencing on IL-10 accumulation levels, transient silencing experiments were performed in tobacco plants that were 1.5 ft tall. A. tumefaciens containing CysP6-Si construct was infiltrated into tobacco leaf epidermal cells and samples were collected for evaluating CysP6 transcript and IL-10 protein accumulation. Our RT-PCR analysis using gene specific primers did not detect any CysP6 transcript in all 7 biological replicates whereas it was present in vector-only plants (Fig 6A). This suggests that CysP6 was efficiently silenced by transient expression of the silencing construct. Furthermore, IL-10 levels were significantly higher in all 7 biological replicates which were infiltrated with the CysP6 silencing construct, than tissues infiltrated with the vector-only constructs (Fig 6B). These results demonstrated that transient silencing CysP6 results in the increase in IL-10 accumulation in G7 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.