Three unrelated protease inhibitors enhance accumulation of pharmaceutical recombinant proteins in Nicotiana benthamiana
Author(s)
Type
Journal Article
Abstract
Agroinfiltrated Nicotiana benthamiana is a flexible and scalable platform for recombinant protein (RP) production, 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 PIs enhancing RP accumulation, we found only cystatin SlCYS8 to be effective. We identified three additional new, unrelated PIs that enhance RP accumulation: N. benthamiana NbPR4, NbPot1 and human HsTIMP, which have been reported to inhibit cysteine, serine and metalloproteases, respectively. Remarkably, accumulation of all three RPs is enhanced by each PI similarly, suggesting that the mechanism of degradation of unrelated RPs follows a common pathway. Inhibitory functions HsTIMP and SlCYS8 are required to enhance RP accumulation, suggesting that 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 activities of papain-like Cys proteases (PLCPs), Ser hydrolases (SHs) or vacuolar processing enzymes (VPEs) in leaves are unaffected upon expression of the new PIs, whereas SlCYS8 expression specifically suppresses PLCP activity only. Quantitative proteomics indicates that the three new PIs affect agroinfiltrated tissues similarly and that they all increase immune responses. NbPR4, NbPot1 and HsTIMP can be used to study plant proteases and improve RP accumulation in molecular farming.
Date Issued
2018-10
Date Acceptance
2018-02-28
Citation
Plant Biotechnology Journal, 2018, 16 (10), pp.1797-1810
ISSN
1467-7644
Publisher
Wiley
Start Page
1797
End Page
1810
Journal / Book Title
Plant Biotechnology Journal
Volume
16
Issue
10
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.
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.
License URL
Identifier
https://www.ncbi.nlm.nih.gov/pubmed/29509983
Subjects
Nicotiana
Plant Immunity
Plant Leaves
Plants, Genetically Modified
Proteinase Inhibitory Proteins, Secretory
Proteolysis
Proteome
Recombinant Proteins
Agrobacterium tumefaciens
Nicotiana benthamiana
activity-based protein profiling
agroinfiltration
protease inhibitor
transient expression
Publication Status
Published
Coverage Spatial
England
Date Publish Online
2018-03-06