Rapid and Scalable Plant-Based Production of a Potent Plasmin Inhibitor Peptide

Abstract

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.

Sunflower Trypsin Inhibitor‐1 (SFTI‐1): Sowing Seeds in the Fields of Chemistry and Biology

“…In parallel with studies exploring the promising applications of SFTI-1, another important area of work relates to developing economic methods for the large-scale production of SFTI-1 and other cyclic peptides.T his objective is highly relevant to the feasibility of future commercial applications, and there have been exciting developments in synthetic, biomimetic,cell-based, and in planta approaches.One example is the successful production of SFTI-1 and several engineered analogues in tobacco,w hich suggests that, with further optimisation, al arge-scale plant-based system might be af easible approach for the production of cyclic peptides. [110] Ab iological approach could also provide am ore eco-friendly and sustainable alternative to traditional chemical synthesis.C ell-based systems,s uch as E. coli,h ave been shown to express cyclic SFTI-1 using inteins,a nd production could also be adapted to build in additional processing steps, including using enzymes to cyclise purified precursors.Overall, we see abright future for SFTI-1 in the fields of chemistry and biology that will nurture the growth of new and existing ideas,u ntil they ultimately blossom into established therapeutics and technologies.…”

“…[109] Arecent study explored the potential of this system for large-scale production, by using an expression construct comprising several tandem repeats of the target peptide. [110] Thea pproach was tested using the engineered plasmin inhibitor 28,a nd co-expression with OaAEP1b resulted in yields of target peptide of up to 60 mgp er gram of leaf dry weight. [110] Plant-based expression systems have huge untapped potential for the production of awide range of high-value therapeutics,i ncluding cyclic peptides,a nd with further optimisation in the future,they may become alow cost alternative to chemical synthesis.…”

A bifunctional asparaginyl endopeptidase efficiently catalyzes both cleavage and cyclization of cyclic trypsin inhibitors

“…MCoAEP2 can cyclize an engineered MCoTI-II scaffold. AEPs (like butelase 1 and OaAEP1 b ) have previously been shown to have a wide range of biotechnological applications, including peptide/protein ligation, peptide macrocyclization, and cell surface labeling [32][33][34][35][36][37] ; however, their utility in grafted peptide production is unclear 38 . As MCoAEP2 can produce native MCoTI-II in vitro, we hypothesized this enzyme could function as an ideal cyclase for grafted peptide production.…”

Der Sonnenblumen‐Trypsin‐Inhibitor 1 (SFTI‐1) in der Chemie und Biologie

“…[109] Eine kürzlich durchgeführte Studie untersuchte das Potenzial dieses Systems fürd ie Produktion in großem Maßstab unter Verwendung eines Expressionskonstrukts,d as mehrere Tandem-Wiederholungen des Zielpeptids umfasst. [110] Der Ansatz wurde unter Verwendung des Plasmininhibitors 28 getestet, und die Coexpression mit OaAEP1b führte zu Ausbeuten von bis zu 60 mg Zielpeptid pro Gramm Blatttrockengewicht. [110] Pflanzliche Expressionssysteme haben ein enormes ungenutztes Potenzial fürd ie Herstellung einer breiten Palette hochwertiger Therapeutika, einschließlich zyklischer Peptide,u nd werden in Zukunft mçglicherweise weiter optimiert werden, um eine kostengünstige Alternative zur chemischen Synthese zu werden.…”

“…Ein Beispiel ist die Herstellung von SFTI-1 und mehreren Analoga in Tabakpflanzen, was darauf hindeutet, dass bei weiterer Optimierung ein pflanzliches Großsystem ein praktikabler Ansatz fürd ie zyklische Peptidproduktion sein kçnnte. [110] Ein biologischer Ansatz kçnnte auch eine umweltfreundlichere und nachhaltigere Alternative zur traditionellen chemischen Synthese darstellen. Es wurde gezeigt, dass zellbasierte Systeme wie E. coli zyklisches SFTI-1 unter Verwendung von Inteinen exprimieren, und die Produktion kçnnte auch angepasst werden, um zusätzliche Verarbeitungsschritte einzubauen, einschließlich der Verwendung von Enzymen zur Zyklisierung aufgereinigter Vorläufer.…”