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Grosse-Holz, Friederike, Madeira, Luisa, Zahid, Muhammad Awais, Songer, Molly, Kourelis, Jiorgos 
ORCID: https://orcid.org/0000-0002-9007-1333, Fesenko, Mary, Ninck, Sabrina, Kaschani, Farnusch, Kaiser, Markus and van der Hoorn, Renier A. L.
(2018)
Three unrelated protease inhibitors enhance accumulation of pharmaceutical recombinant proteins in Nicotiana benthamiana.
Plant Biotechnology Journal, 16 (10).
pp. 1797-1810.
ISSN 1467-7644
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.
| Item Type: | Article |
|---|---|
| Additional Information: | Funding Information: This work was financially supported by the ERC Project ‘GreenProteases’, University of Oxford (RH, grant No. 616449), by Somerville College, Oxford (FGH and RH), an ERC starting grant (MK, grant No. 258413) and the Deutsche Forschungsgemeinschaft (MK, grant no. INST 20876/127-1 FUGG). MAZ was supported by an Erasmus grant, MS was supported by the BBSRC’s Research Experience Placements scheme and MF was supported by the David Kirby Memorial Fund. |
| Uncontrolled Keywords: | activity-based protein profiling,agrobacterium tumefaciens,agroinfiltration,nicotiana benthamiana,protease inhibitor,transient expression,biotechnology,agronomy and crop science,plant science ,/dk/atira/pure/subjectarea/asjc/1300/1305 |
| Faculty \ School: | Faculty of Science > The Sainsbury Laboratory |
| Related URLs: | |
| Depositing User: | LivePure Connector |
| Date Deposited: | 07 Dec 2023 01:46 |
| Last Modified: | 01 Feb 2024 03:08 |
| URI: | https://ueaeprints.uea.ac.uk/id/eprint/93900 |
| DOI: | 10.1111/pbi.12916 |
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