Three unrelated protease inhibitors enhance accumulation of pharmaceutical recombinant proteins in N. benthamiana

ABSTRACT: Agroinfiltrated Nicotiana benthamiana is a flexible and scalable recombinant protein (RP) production platform, but its great potential is hampered by plant proteases that degrade RPs. Here, we tested 29 candidate protease inhibitors (PIs) in agroinfiltrated N. benthamiana leaves for enhancing accumulation of three unrelated RPs: glycoenzyme α-Galactosidase, glycohormone erythropoietin (EPO) and IgG antibody VRC01. Of the previously described RP accumulation enhancing PIs, we found the cystatin SlCYS8 to be effective. We identified three additional new, unrelated PIs that enhanced RP accumulation: N. benthamiana NbPR4, NbPot1 and human HsTIMP, which are described to inhibit cysteine, serine and metalloproteases, respectively. Remarkably, accumulation of three unrelated RPs is enhanced by each PI, suggesting the mechanism of RP degradation may contain universal elements. Inhibitory motifs of HsTIMP and SlCYS8 are required to enhance RP accumulation, suggesting their target proteases may degrade RPs. Different PIs additively enhance RP accumulation, but the effect of each PI is dose-dependent. Activity-based Protein Profiling (ABPP) revealed that the activity profiles of Papain-like Cys proteases (PLCPs), Ser hydrolases (SHs) or Vacuolar Processing Enzymes (VPEs) in leaves are unaffected by the new PIs, whereas SlCYS8 specifically only suppresses PLCP activity. Quantitative leaf proteome data indicate that the three new PIs affect agroinfiltrated tissues similarly and that they all increase plant immunity. Our data indicate that RPs are degraded stepwise by a redundant, dynamic network of plant proteases. NbPR4, NbPot1 and HsTIMP can be used to identify and control new plant proteases and to enhance RP accumulation in industrial molecular farming.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Agrobacterium Fabrum Str. C58 Nicotiana Benthamiana

TISSUE(S): Plant Cell, Leaf

SUBMITTER: Farnusch Kaschani  

LAB HEAD: Farnusch Kaschani

PROVIDER: PXD008538 | Pride | 2018-05-17

REPOSITORIES: Pride