Expression, purification and functional validation of a cancer-associated isoform of the HBx protein from human hepatitis B virus

The human hepatitis B virus (HBV) causes hepatitis B, a liver infection that can be acute or chronic. HBV encodes four proteins, among which the X protein (HBx) plays a critical role in viral replication. During chronic HBV infection, in which the viral DNA is integrated into the host genome, the HBx 1-120 isoform, comprising the N-terminal 120 residues, is highly expressed. Here, we describe a protocol for the recombinant overexpression and purification of untagged HBx 1-120 from bacterial cells. The procedure is compatible with stable isotope labelling in minimal media. Following cell lysis, HBx 1-120 was recovered from inclusion bodies (IBs), solubilized in urea, and purified by ion-exchange (IEX) and size-exclusion chromatography (SEC). The purified protein was extensively characterized, including by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. Functionality was confirmed by a pulldown assay with a known interacting partner, Spindlin1. This protocol provides a robust framework to obtain untagged HBx 1-120 for structural and functional in vitro studies. • Protocol for expression and purification of HBx protein from human Hepatitis B virus • Native protein sequence (isoform HBx 1-120 ) without tags or modifications • Protein stable and monodisperse at high micromolar concentrations for biophysical and structural studies • Compatible with stable isotope labelling in minimal media