Grafting Epitopes into a Disulfide-Constrained Tail of a Lasso Peptide

Abstract

Bioactive peptides are a promising direction for drug development. The specific goal of this study was to add a bioactive sequence to the C-terminus of the lasso peptide astexin-3 (Atx-3). Two sequences were tested, both containing the motif WEWEC. This motif inhibits activity of vascular endothelial growth factor, VEGF, an extracellular ligand produced in excess by cancer cells. The results of this study use mass spectroscopy to show the potential for adding a bioactive sequence to the C-terminus of Atx-3. The first sequence (WEWEC), called short-WEWEC, was successfully inserted into an existing Atx-3 vector and induced to produce varying levels of the full-length peptide. The lower temperature (16 degrees C) produced relatively higher signals of the full-length peptide at 3227 m/z and decreased C-terminus truncation. A longer induction time (48 hours) was needed for peptide production and for disulfide bond formation. The long-WEWEC sequence (IHVMWEWEC) was also successfully inserted into an existing Atx-3 vector. Similarly, a lower temperature (16 degrees C) and longer times of induction resulted in potential dimerization and decreased truncation of the full-length peptide. Although the full-length long-WEWEC peptide (Atx-3 with IHVMWEWEC addition to C-terminus) wasn’t observed, it appears the truncation intermediates favored three or four additional residues at the tail of the original Atx-3 peptide.