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Three unrelated protease inhibitors enhance accumulation of pharmaceutical recombinant proteins in Nicotiana benthamiana
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 ac...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
John Wiley and Sons Inc.
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6131417/ https://www.ncbi.nlm.nih.gov/pubmed/29509983 http://dx.doi.org/10.1111/pbi.12916 |
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author | Grosse‐Holz, Friederike Madeira, Luisa Zahid, Muhammad Awais Songer, Molly Kourelis, Jiorgos Fesenko, Mary Ninck, Sabrina Kaschani, Farnusch Kaiser, Markus van der Hoorn, Renier A.L. |
author_facet | Grosse‐Holz, Friederike Madeira, Luisa Zahid, Muhammad Awais Songer, Molly Kourelis, Jiorgos Fesenko, Mary Ninck, Sabrina Kaschani, Farnusch Kaiser, Markus van der Hoorn, Renier A.L. |
author_sort | Grosse‐Holz, Friederike |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-6131417 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | John Wiley and Sons Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-61314172018-09-13 Three unrelated protease inhibitors enhance accumulation of pharmaceutical recombinant proteins in Nicotiana benthamiana Grosse‐Holz, Friederike Madeira, Luisa Zahid, Muhammad Awais Songer, Molly Kourelis, Jiorgos Fesenko, Mary Ninck, Sabrina Kaschani, Farnusch Kaiser, Markus van der Hoorn, Renier A.L. Plant Biotechnol J Research Articles 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. John Wiley and Sons Inc. 2018-05-24 2018-10 /pmc/articles/PMC6131417/ /pubmed/29509983 http://dx.doi.org/10.1111/pbi.12916 Text en © 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 http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Articles Grosse‐Holz, Friederike Madeira, Luisa Zahid, Muhammad Awais Songer, Molly Kourelis, Jiorgos Fesenko, Mary Ninck, Sabrina Kaschani, Farnusch Kaiser, Markus van der Hoorn, Renier A.L. Three unrelated protease inhibitors enhance accumulation of pharmaceutical recombinant proteins in Nicotiana benthamiana |
title | Three unrelated protease inhibitors enhance accumulation of pharmaceutical recombinant proteins in Nicotiana benthamiana
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title_full | Three unrelated protease inhibitors enhance accumulation of pharmaceutical recombinant proteins in Nicotiana benthamiana
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title_fullStr | Three unrelated protease inhibitors enhance accumulation of pharmaceutical recombinant proteins in Nicotiana benthamiana
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title_full_unstemmed | Three unrelated protease inhibitors enhance accumulation of pharmaceutical recombinant proteins in Nicotiana benthamiana
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title_short | Three unrelated protease inhibitors enhance accumulation of pharmaceutical recombinant proteins in Nicotiana benthamiana
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title_sort | three unrelated protease inhibitors enhance accumulation of pharmaceutical recombinant proteins in nicotiana benthamiana |
topic | Research Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6131417/ https://www.ncbi.nlm.nih.gov/pubmed/29509983 http://dx.doi.org/10.1111/pbi.12916 |
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