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  • Recombinant protein susceptibility to proteolysis in the plant cell secretory pathway is pH-dependent

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    SAINSBURY221469.pdf (851.9Kb)
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    Author(s)
    Jutras, Philippe V
    Goulet, Marie-Claire
    Lavoie, Pierre-Olivier
    D'Aoust, Marc-Andre
    Sainsbury, Frank
    Michaud, Dominique
    Griffith University Author(s)
    Sainsbury, Frank
    Year published
    2018
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    Abstract
    Cellular engineering approaches have been proposed to mitigate unintended proteolysis in plant protein biofactories, involving the design of protease activity‐depleted environments by gene silencing or in situ inactivation with accessory protease inhibitors. Here, we assessed the impact of influenza virus M2 proton channel on host protease activities and recombinant protein processing in the cell secretory pathway of Nicotiana benthamiana leaves. Transient co‐expression assays with M2 and GFP variant pHluorin were first conducted to illustrate the potential of proton export from the Golgi lumen to promote recombinant protein ...
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    Cellular engineering approaches have been proposed to mitigate unintended proteolysis in plant protein biofactories, involving the design of protease activity‐depleted environments by gene silencing or in situ inactivation with accessory protease inhibitors. Here, we assessed the impact of influenza virus M2 proton channel on host protease activities and recombinant protein processing in the cell secretory pathway of Nicotiana benthamiana leaves. Transient co‐expression assays with M2 and GFP variant pHluorin were first conducted to illustrate the potential of proton export from the Golgi lumen to promote recombinant protein yield. A fusion protein‐based system involving protease‐sensitive peptide linkers to attach inactive variants of tomato cystatin SlCYS8 was then designed to relate the effects of M2 on protein levels with altered protease activities in situ. Secreted versions of the cystatin fusions transiently expressed in leaf tissue showed variable ‘fusion to free cystatin’ cleavage ratios, in line with the occurrence of protease forms differentially active against the peptide linkers in the secretory pathway. Variable ratios were also observed for the fusions co‐expressed with M2, but the extent of fusion cleavage was changed for several fusions, positively or negatively, as a result of pH increase in the Golgi. These data indicating a remodelling of endogenous protease activities upon M2 expression confirm that the stability of recombinant proteins in the plant cell secretory pathway is pH‐dependent. They suggest, in practice, the potential of M2 proton channel to modulate the stability of protease‐susceptible secreted proteins in planta via a pH‐related, indirect effect on host resident proteases.
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    Journal Title
    PLANT BIOTECHNOLOGY JOURNAL
    Volume
    16
    Issue
    11
    DOI
    https://doi.org/10.1111/pbi.12928
    Copyright Statement
    © 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
    Subject
    Biological sciences
    Biomedical and clinical sciences
    Publication URI
    http://hdl.handle.net/10072/385706
    Collection
    • Journal articles

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