# Protocol for producing brain-derived neurotrophic factor and neurotrophin-4 in their pro and active form in Escherichia coli

## TL;DR

This paper provides a detailed protocol for producing brain-derived neurotrophic factor and neurotrophin-4 in both their inactive and active forms using E. coli.

## Contribution

The paper introduces a reproducible method for producing pro and active forms of BDNF and NT4 in E. coli with proper disulfide bridges.

## Key findings

- A protocol for producing proBDNF and proNT4 in E. coli is described.
- Steps for oxidative in vitro folding and purification of the neurotrophins are detailed.
- Proteolytic activation and characterization methods are provided.

## Abstract

Brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT4) are neurotrophic growth factors, signaling primarily via the tropomyosin receptor kinase B (TrkB). Here, we present a protocol for producing BDNF and NT4 in their pro and active form in Escherichia coli. We describe steps for E. coli expression, oxidative in vitro folding from inclusion bodies, and purification. We then detail procedures for proteolytic activation and characterization of the purified neurotrophins.

For complete details on the use and execution of this protocol, please refer to Holzner et al.1

•Instructions for producing proBDNF and proNT4 inclusion bodies in E. coli•Steps for in vitro folding of proBDNF and proNT4 with intact disulfide bridges•Guidance on batch purification and activation of folded proBDNF and proNT4

Instructions for producing proBDNF and proNT4 inclusion bodies in E. coli

Steps for in vitro folding of proBDNF and proNT4 with intact disulfide bridges

Guidance on batch purification and activation of folded proBDNF and proNT4

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

Brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT4) are neurotrophic growth factors, signaling primarily via the tropomyosin receptor kinase B (TrkB). Here, we present a protocol for producing BDNF and NT4 in their pro and active form in Escherichia coli. We describe steps for E. coli expression, oxidative in vitro folding from inclusion bodies, and purification. We then detail procedures for proteolytic activation and characterization of the purified neurotrophins.