# Homologous expression and purification of human HAX‐1 for structural studies

## TL;DR

This paper describes a method to produce and purify human HAX-1 protein for studying its structure and interactions.

## Contribution

A new protocol for expressing and purifying HAX-1 with a cleavable GFP tag in mammalian cells.

## Key findings

- The protocol yields high amounts of soluble HAX-1 protein.
- The method enables structural and interaction studies of HAX-1.
- HAX-1 is predicted to be disordered without a binding partner.

## Abstract

The human HAX‐1 protein is a ubiquitously expressed multifunctional protein that regulates various cellular processes through interactions with numerous cellular proteins and RNA. The purification protocol presented here is designed explicitly for HAX‐1 expressed in mammalian cells and yields high amounts of soluble HAX‐1 protein constructs N‐terminally fused to a cleavable superfolder GFP. This protocol will enable structural studies of HAX‐1, which is predicted to undergo posttranslational modifications and be partially disordered in the absence of a binding partner.

This research protocol provides detailed instructions for cloning, expressing, and purifying large quantities of the intrinsically disordered human HAX‐1 protein, N‐terminally fused to a cleavable superfolder GFP, from mammalian cells. HAX‐1 is predicted to undergo posttranslational modifications and to interact with membranes, various cellular proteins, viral proteins, and RNA. This protocol aims to facilitate in vitro protein–protein interaction studies and structural analyses.