# Exploring Use of a Protein Cage System for Producing Bioactive Peptides in Escherichia coli

## TL;DR

This paper explores using virus-like particles in E. coli to produce bioactive peptides, which could lead to new treatments for drug-resistant diseases.

## Contribution

The study introduces a novel method for encapsulating bioactive peptides using a P22 virus-like particle system in E. coli.

## Key findings

- BAPs were successfully encapsulated into P22 VLPs at high ratios.
- Encapsulation improved purification and functional equivalence of produced peptides.
- Co-expression of CP did not enhance BAP yields despite high loading densities.

## Abstract

New therapeutics are urgently needed to curb the spread of drug‐resistant diseases. Bioactive peptides (BAPs), including antimicrobial peptides, are emerging as an exciting new class of compounds with advantages over current drug modalities, especially small molecule drugs that are prone to resistance development. Here, we evaluated a bacteriophage P22 virus‐like particle (VLP) system where BAPs are encapsulated as fusion proteins with the P22 scaffold protein (SP) within self‐assembling protein cages in
Escherichia coli
. Representative peptides from three structurally distinct classes of BAPs were successfully encapsulated into P22 VLPs at high cargo to VLP coat protein (CP) ratios that corresponded to interactions between the compact electropositive structures of the SP‐BAPs and electronegative regions on the inward facing surface of CP subunits. However, high loading densities did not correspond to improved SP‐BAP yields. An unexpected finding of this study was that while encapsulation alleviated negative effects of SP‐BAPs on
E. coli
growth, the P22 scaffold protein acted as a sufficient fusion partner for accumulating BAPs, and co‐expression of the CP did not further improve SP‐BAP yields. Nevertheless, encapsulation in VLPs provided a useful first step in the purification pipeline for producing both linear and cyclic recombinant (r)BAPs that were functionally equivalent to their synthetic counterparts. Further efforts to optimise expression ratios of CP to SP‐BAP fusions will be required to realise the full potential of encapsulation for protecting expression hosts and maximising rBAP yields.

Bioactive peptides can be difficult to manufacture biosynthetically. This study explored the use of bacteriophage P22 virus‐like particles to encapsulate recombinantly expressed bioactive peptides, enabling the production of therapeutically valuable linear and cyclic peptides in
Escherichia coli
.