Scalable and Efficient In Planta Biosynthesis of Sunflower Trypsin Inhibitor-1 (SFTI) Peptide Therapeutics.

Sunflower trypsin inhibitor-1 (SFTI-1) is a 14 amino acid cyclic peptide which has been effectively employed as a scaffold for engineering a range of peptide therapeutic candidates. Typically, synthesis of SFTI-1-based therapeutics is performed via solid-phase peptide synthesis and native chemical ligation, with significant financial and environmental costs associated. In planta synthesis of SFTI-1 based therapeutics serves as a greener approach for environmentally sustainable production. Here, we detail the methods for the transient expression, production, and purification of SFTI-1-based therapeutic peptides in Nicotiana benthamiana using a scalable and high-throughput approach. We demonstrate that a prerequisite for this is the co-expression of specialized asparaginyl endopeptidases (AEPs) that perform the backbone cyclization of SFTI-1. In our founding study, we were able to achieve in planta yields of a plasmin inhibitor SFTI-1 peptide at yields of ~60 μg/g of dried plant material.