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

Philippe V Jutras,Dominique Michaud,Marie‐Claire Goulet,Pierre‐Olivier Lavoie,Frank Sainsbury,Marc‐André D'Aoust

doi:10.1111/pbi.12928

Philippe V Jutras, Dominique Michaud + Show 4 more

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https://doi.org/10.1111/pbi.12928

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Abstract

SummaryCellular 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.

Highlights

The value of plants as biological factories for medically useful proteins has been confirmed in recent years with the approval of a first plant-made biopharmaceutical for human therapy, the successful production of therapeutic antibodies in plants during the recent Ebola virus outbreak and the increasing number of plant-derived proteins subject to clinical trials for human use (Lomonossoff and D’Aoust, 2016; Ma et al, 2015; Sack et al, 2015)
Fluorescence was detected in leaves infiltrated to co-express pHluorin and M2, at a relative intensity two to three times the intensity measured in leaves expressing pHluorin alone
This promoting effect of M2 on pHluorin content was not observed with A30P, an inactive mutant of M2 properly expressed in N. benthamiana leaves (Jutras et al, 2015) but unable to drive proton transport out of the secretory pathway (Holsinger et al, 1994)

Summary

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. Variable ratios were 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 pHdependent. 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

Introduction

Results and discussion

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Conclusion

Experimental procedures