title: Identification, Characterization and Down-Regulation of Cysteine Protease Genes in Tobacco for Use in Recombinant Protein Production.
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.
authors.affiliation: Department of Biology, University of Western Ontario, London, ON, Canada.
authors.forename: Kishor
authors.initials: K
authors.lastname: Duwadi
authors.affiliation: Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada.
authors.forename: Ling
authors.initials: L
authors.lastname: Chen
authors.affiliation: Department of Biology, University of Western Ontario, London, ON, Canada; Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada.
authors.forename: Rima
authors.initials: R
authors.lastname: Menassa
authors.affiliation: Department of Biology, University of Western Ontario, London, ON, Canada; Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada.
authors.forename: Sangeeta
authors.initials: S
authors.lastname: Dhaubhadel
mesh_terms: D057056:Cysteine Proteases / Q000235:genetics*; D015536:Down-Regulation / Q000235:genetics*; D004721:Endoplasmic Reticulum / Q000235:genetics; D018506:Gene Expression Regulation, Plant / Q000235:genetics*; D006801:Humans; D016753:Interleukin-10 / Q000235:genetics; D018515:Plant Leaves / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics; D030821:Plants, Genetically Modified / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics*; D014026:Nicotiana / Q000235:genetics*
chemical_list: C508609:IL10 protein, human; D010940:Plant Proteins; D011994:Recombinant Proteins; D016753:Interleukin-10; D057056:Cysteine Proteases