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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.


Schematic representation of different domains in papain and otubain family CysPs.Candidate CysPs having specific domain types are indicated. The diagram is not drawn to scale. I29, Cathepsin propeptide inhibitor domain; Peptidase_C1, Papain family domain; GRN, Granulin like repeats; Peptidase_C65, Otubain family domain.
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pone.0130556.g001: Schematic representation of different domains in papain and otubain family CysPs.Candidate CysPs having specific domain types are indicated. The diagram is not drawn to scale. I29, Cathepsin propeptide inhibitor domain; Peptidase_C1, Papain family domain; GRN, Granulin like repeats; Peptidase_C65, Otubain family domain.

Mentions: To determine the domain composition of the candidate CysP proteins in [32] tobacco, we performed the domain analysis using pfam database (http://pfam.xfam.org/). The analysis revealed that CysP1-CysP8 and CysP10 each contain a common papain domain, shown to be responsible for the endo-peptidase activity of CysPs. Proteins containing such domains are classified under the papain or peptidase_C1 family and are synthesized as precursor CysPs [39,40]. Precursor CysPs have an additional propeptide sequence preceding their papain domain, called I29 or the cathepsin propeptide inhibitor domain (Fig 1). CysP6 and CysP8 have an additional granulin-like repeat that is known to play a role in targeting and activity regulation of certain CysPs [41]. CysP9 contains a single large ovarian tumor (OTU) domain suggesting it may belong to the otubain or peptidase_C65, and possibly possess functions similar to deubiquitinating enzymes of the peptidase_C65 family. Detailed information regarding putative domain structure of all candidate CysPs is shown in Table 3.


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)

Schematic representation of different domains in papain and otubain family CysPs.Candidate CysPs having specific domain types are indicated. The diagram is not drawn to scale. I29, Cathepsin propeptide inhibitor domain; Peptidase_C1, Papain family domain; GRN, Granulin like repeats; Peptidase_C65, Otubain family domain.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130556.g001: Schematic representation of different domains in papain and otubain family CysPs.Candidate CysPs having specific domain types are indicated. The diagram is not drawn to scale. I29, Cathepsin propeptide inhibitor domain; Peptidase_C1, Papain family domain; GRN, Granulin like repeats; Peptidase_C65, Otubain family domain.
Mentions: To determine the domain composition of the candidate CysP proteins in [32] tobacco, we performed the domain analysis using pfam database (http://pfam.xfam.org/). The analysis revealed that CysP1-CysP8 and CysP10 each contain a common papain domain, shown to be responsible for the endo-peptidase activity of CysPs. Proteins containing such domains are classified under the papain or peptidase_C1 family and are synthesized as precursor CysPs [39,40]. Precursor CysPs have an additional propeptide sequence preceding their papain domain, called I29 or the cathepsin propeptide inhibitor domain (Fig 1). CysP6 and CysP8 have an additional granulin-like repeat that is known to play a role in targeting and activity regulation of certain CysPs [41]. CysP9 contains a single large ovarian tumor (OTU) domain suggesting it may belong to the otubain or peptidase_C65, and possibly possess functions similar to deubiquitinating enzymes of the peptidase_C65 family. Detailed information regarding putative domain structure of all candidate CysPs is shown in Table 3.

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.