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

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.