A protease activity-depleted environment for heterologous proteins migrating towards the leaf cell apoplast

Goulet, Charles, Khalf, Moustafa, Sainsbury, Frank, D'Aoust, Marc-Andre and Michaud, Dominique (2012) A protease activity-depleted environment for heterologous proteins migrating towards the leaf cell apoplast. Plant Biotechnology Journal, 10 1: 83-94. doi:10.1111/j.1467-7652.2011.00643.x

Author Goulet, Charles
Khalf, Moustafa
Sainsbury, Frank
D'Aoust, Marc-Andre
Michaud, Dominique
Title A protease activity-depleted environment for heterologous proteins migrating towards the leaf cell apoplast
Journal name Plant Biotechnology Journal   Check publisher's open access policy
ISSN 1467-7644
Publication date 2012-01-01
Year available 2012
Sub-type Article (original research)
DOI 10.1111/j.1467-7652.2011.00643.x
Open Access Status DOI
Volume 10
Issue 1
Start page 83
End page 94
Total pages 12
Place of publication Oxford, United Kingdom
Publisher Wiley-Blackwell Publishing Ltd.
Language eng
Formatted abstract
 Recombinant proteins face major constraints along the plant cell secretory pathway, including proteolytic processing compromising their structural integrity. Here, we demonstrate the potential of protease inhibitors as in situ stabilizing agents for recombinant proteins migrating towards the leaf apoplast. Genomic data for Arabidopsis, rice and Nicotiana spp. were assessed to determine the relative incidence of protease families in the cell secretory pathway. Transient expression assays with the model platform Nicotiana benthamiana were then performed to test the efficiency of protease inhibitors in stabilizing proteins targeted to the apoplast. Current genomic data suggest the occurrence of proteases from several families along the secretory pathway, including A1 and A22 Asp proteases; C1A and C13 Cys proteases; and S1, S8 and S10 Ser proteases. In vitro protease assays confirmed the presence of various proteases in N. benthamiana leaves, notably pointing to the deposition of A1- and S1-type activities preferentially in the apoplast. Accordingly, transient expression and secretion of the A1/S1 protease inhibitor, tomato cathepsin D inhibitor (SlCDI), negatively altered A1 and S1 protease activities in this cell compartment, while increasing the leaf apoplast protein content by ∼45% and improving the accumulation of a murine diagnostic antibody, C5-1, co-secreted in the apoplast. SlCYS9, an inhibitor of C1A and C13 Cys proteases, had no impact on the apoplast proteases and protein content, but stabilized C5-1 in planta, presumably upstream in the secretory pathway. These data confirm, overall, the potential of protease inhibitors for the in situ protection of recombinant proteins along the plant cell secretory pathway.
Keyword Cell secretory pathway
Leaf apoplast proteome
Molecular farming
Nicotiana benthamiana
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ

Document type: Journal Article
Sub-type: Article (original research)
Collection: Australian Institute for Bioengineering and Nanotechnology Publications
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Citation counts: TR Web of Science Citation Count  Cited 37 times in Thomson Reuters Web of Science Article | Citations
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