Construction of Lasso Peptide Fusion Proteins

Abstract

Lasso peptides are a family of ribosomally-synthesized and posttranslationally-modified peptides (RiPPs) typified by an isopeptide-bonded macrocycle between the peptide N-terminus and an aspartate or glutamate sidechain. The C-terminal portion of the peptide threads through the N-terminal macrocycle to give the characteristic lasso fold. Because of the inherent stability, both proteolytic and often thermal, of lasso peptides, we became interested in whether proteins could be fused to the free C-terminus of lasso peptides. Here we demonstrate fusion of two model proteins, the artificial leucine zipper A1 and the superfolder variant of GFP, to the C-terminus of the lasso peptide astexin-1. Successful lasso cyclization of the N-terminus of these fusion proteins requires a flexible linker in between the C-terminus of the lasso peptide and the N-terminus of the protein of interest. The ability to fuse lasso peptides to a protein of interest is an important step toward phage and bacterial display systems for the high-throughput screening of lasso peptide libraries for new functions.

The B1 Protein Guides the Biosynthesis of a Lasso Peptide

“…Such discrepancies may be explained by the evolutionary distance between the two systems; i.e., they originate from different phyla, contain distinct gene cluster arrangements, and differ in B protein composition (PadeB is split, MjcB is fused). Additionally, a recent study provided experimental evidence that the B and C proteins of the astexin-1 system may also function independently of one another 52 . Hence, our results indicate that not all lasso peptide processing enzymes are necessarily interdependent like McjB and McjC.…”

Lasso Peptides: Heterologous Production and Potential Medical Application

“…In addition, it is possible to generate lasso peptide-protein fusions by joining the end of a lasso peptide precursor to a protein and co-expressing it with the processing enzymes. In this way, Link and co-workers accomplished the generation of fusions of mature astexin-1 lasso peptide to the N-terminus of GFP and a leucin zipper protein ( Zong et al, 2016 ). Furthermore, the chemically-synthesized macrolactam ring from RES-701-1 was coupled to some bioactive peptides, thereby were combining activities and enhancing stabilities ( Shibata et al, 2003 ).…”

“…Lasso peptides are suitable molecular backbones for epitope grafting, owing to their thermal and proteolytic stability ( Hegemann, 2019 ). Modified lasso peptides could be applied as molecular probes or as drug carries for therapeutic applications ( Knappe et al, 2011 ; Piscotta et al, 2015 ; Zong et al, 2016 ). However, these modifications also lead to lowered production levels ( Zimmermann et al, 2013 , 2014 ; Hegemann et al, 2016 ; Zong et al, 2017 ).…”

Reactivity-based screening for citrulline-containing natural products reveals a family of bacterial peptidyl arginine deiminases

“…4 These properties, in addition to the substrate permissiveness of several characterized lasso biosynthetic enzymes, have engendered interest in the design of lasso peptides therapeutics. 5,6,7,8,9 After ribosomal synthesis of the lasso precursor peptide, the next biosynthetic step involves RiPP recognition element (RRE) engagement of the precursor peptide, which occurs through binding to a recognition sequence within the Nterminal leader region. The RRE domain mediates recruitment of the subsequent enzymes 10,11 and is found as either a discretely encoded protein or fused to the leader peptidase.…”