Rapid and Scalable Plant-Based Production of a Potent Plasmin Inhibitor Peptide
The backbone cyclic and disulfide bridged sunflower trypsin inhibitor-1 (SFTI-1) peptide is a proven effective scaffold for a range of peptide therapeutics. For production at laboratory scale, solid phase peptide synthesis techniques are widely used, but these synthetic approaches are costly and env...
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doaj-1cf8e40b56734239a099631164e722fb2020-11-24T21:44:25ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2019-05-011010.3389/fpls.2019.00602441863Rapid and Scalable Plant-Based Production of a Potent Plasmin Inhibitor PeptideMark A. Jackson0Kuok Yap1Aaron G. Poth2Edward K. Gilding3Joakim E. Swedberg4Simon Poon5Haiou Qu6Thomas Durek7Karen Harris8Marilyn A. Anderson9David J. Craik10Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, AustraliaInstitute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, AustraliaInstitute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, AustraliaInstitute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, AustraliaInstitute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, AustraliaDepartment of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, AustraliaInstitute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, AustraliaInstitute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, AustraliaDepartment of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, AustraliaDepartment of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, AustraliaInstitute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, AustraliaThe backbone cyclic and disulfide bridged sunflower trypsin inhibitor-1 (SFTI-1) peptide is a proven effective scaffold for a range of peptide therapeutics. For production at laboratory scale, solid phase peptide synthesis techniques are widely used, but these synthetic approaches are costly and environmentally taxing at large scale. Here, we developed a plant-based approach for the recombinant production of SFTI-1-based peptide drugs. We show that transient expression in Nicotiana benthamiana allows for rapid peptide production, provided that asparaginyl endopeptidase enzymes with peptide-ligase functionality are co-expressed with the substrate peptide gene. Without co-expression, no target cyclic peptides are detected, reflecting rapid in planta degradation of non-cyclized substrate. We test this recombinant production system by expressing a SFTI-1-based therapeutic candidate that displays potent and selective inhibition of human plasmin. By using an innovative multi-unit peptide expression cassette, we show that in planta yields reach ~60 μg/g dry weight at 6 days post leaf infiltration. Using nuclear magnetic resonance structural analysis and functional in vitro assays, we demonstrate the equivalence of plant and synthetically derived plasmin inhibitor peptide. The methods and insights gained in this study provide opportunities for the large scale, cost effective production of SFTI-1-based therapeutics.https://www.frontiersin.org/article/10.3389/fpls.2019.00602/fullpeptidetherapeuticasparaginyl endopeptidasecyclyzationstabilitybiofactory |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Mark A. Jackson Kuok Yap Aaron G. Poth Edward K. Gilding Joakim E. Swedberg Simon Poon Haiou Qu Thomas Durek Karen Harris Marilyn A. Anderson David J. Craik |
spellingShingle |
Mark A. Jackson Kuok Yap Aaron G. Poth Edward K. Gilding Joakim E. Swedberg Simon Poon Haiou Qu Thomas Durek Karen Harris Marilyn A. Anderson David J. Craik Rapid and Scalable Plant-Based Production of a Potent Plasmin Inhibitor Peptide Frontiers in Plant Science peptide therapeutic asparaginyl endopeptidase cyclyzation stability biofactory |
author_facet |
Mark A. Jackson Kuok Yap Aaron G. Poth Edward K. Gilding Joakim E. Swedberg Simon Poon Haiou Qu Thomas Durek Karen Harris Marilyn A. Anderson David J. Craik |
author_sort |
Mark A. Jackson |
title |
Rapid and Scalable Plant-Based Production of a Potent Plasmin Inhibitor Peptide |
title_short |
Rapid and Scalable Plant-Based Production of a Potent Plasmin Inhibitor Peptide |
title_full |
Rapid and Scalable Plant-Based Production of a Potent Plasmin Inhibitor Peptide |
title_fullStr |
Rapid and Scalable Plant-Based Production of a Potent Plasmin Inhibitor Peptide |
title_full_unstemmed |
Rapid and Scalable Plant-Based Production of a Potent Plasmin Inhibitor Peptide |
title_sort |
rapid and scalable plant-based production of a potent plasmin inhibitor peptide |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Plant Science |
issn |
1664-462X |
publishDate |
2019-05-01 |
description |
The backbone cyclic and disulfide bridged sunflower trypsin inhibitor-1 (SFTI-1) peptide is a proven effective scaffold for a range of peptide therapeutics. For production at laboratory scale, solid phase peptide synthesis techniques are widely used, but these synthetic approaches are costly and environmentally taxing at large scale. Here, we developed a plant-based approach for the recombinant production of SFTI-1-based peptide drugs. We show that transient expression in Nicotiana benthamiana allows for rapid peptide production, provided that asparaginyl endopeptidase enzymes with peptide-ligase functionality are co-expressed with the substrate peptide gene. Without co-expression, no target cyclic peptides are detected, reflecting rapid in planta degradation of non-cyclized substrate. We test this recombinant production system by expressing a SFTI-1-based therapeutic candidate that displays potent and selective inhibition of human plasmin. By using an innovative multi-unit peptide expression cassette, we show that in planta yields reach ~60 μg/g dry weight at 6 days post leaf infiltration. Using nuclear magnetic resonance structural analysis and functional in vitro assays, we demonstrate the equivalence of plant and synthetically derived plasmin inhibitor peptide. The methods and insights gained in this study provide opportunities for the large scale, cost effective production of SFTI-1-based therapeutics. |
topic |
peptide therapeutic asparaginyl endopeptidase cyclyzation stability biofactory |
url |
https://www.frontiersin.org/article/10.3389/fpls.2019.00602/full |
work_keys_str_mv |
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