# Protocol for the recombinant expression and purification of the LSAM domain of human legumain in E. coli

## TL;DR

This paper introduces a new method to express and purify disulfide-rich proteins in E. coli using a fusion tag, demonstrated with the LSAM domain of human legumain.

## Contribution

A novel protocol using the PC1 prodomain fusion tag to express and purify disulfide-containing proteins in E. coli is introduced.

## Key findings

- The PC1 prodomain fusion tag enables expression of disulfide-rich proteins in E. coli.
- The LSAM domain was successfully purified and characterized for structural integrity.
- The protocol allows functional refolding of proteins from non-classical inclusion bodies.

## Abstract

Expressing disulfide-rich proteins in E. coli is challenging due to incorrect bond formation. Here, we present a protocol for expressing the PC1pro-LSAM fusion protein in E. coli using the PC1 prodomain as a fusion tag and the legumain stabilization and activity modulation (LSAM) domain as a proof-of-concept target that was purified. We describe steps for characterizing the protein’s structural integrity through multiple biochemical and biophysical parameters like molecular weight, melting temperature, and secondary structure content. This protocol enables efficient expression of disulfide-containing proteins previously incompatible with bacterial systems.

•PC1 prodomain drives expression in E. coli to non-classical inclusion bodies•PC1-pro fusion tag facilitates protein refolding from non-classical inclusion bodies•Protocol for the preparation of functional, disulfide-linked LSAM domain•Instructions for the expression of disulfide-bonded proteins in E. coli

PC1 prodomain drives expression in E. coli to non-classical inclusion bodies

PC1-pro fusion tag facilitates protein refolding from non-classical inclusion bodies

Protocol for the preparation of functional, disulfide-linked LSAM domain

Instructions for the expression of disulfide-bonded proteins in E. coli

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Expressing disulfide-rich proteins in E. coli is challenging due to incorrect bond formation. Here, we present a protocol for expressing the PC1pro-LSAM fusion protein in E. coli using the PC1 prodomain as a fusion tag and the legumain stabilization and activity modulation (LSAM) domain as a proof-of-concept target that was purified. We describe steps for characterizing the protein’s structural integrity through multiple biochemical and biophysical parameters like molecular weight, melting temperature, and secondary structure content. This protocol enables efficient expression of disulfide-containing proteins previously incompatible with bacterial systems.