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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 HB...

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Main Authors: Clavier, Alexis (Author) , Gómez-Evain, Santiago (Author) , Shida, Toshinobu (Author) , Abanti, Rubaba R. (Author) , Hammerstein, Franziska (Author) , Kielkowski, Pavel (Author) , Leuthold, Mila (Author) , Schütz, Anne (Author)
Format: Article (Journal)
Language:English
Published: February 2026
In: Protein expression and purification
Year: 2026, Volume: 238, Pages: 1-7
ISSN:1096-0279
DOI:10.1016/j.pep.2025.106842
Online Access:Verlag, kostenfrei, Volltext: https://doi.org/10.1016/j.pep.2025.106842
Verlag, kostenfrei, Volltext: https://www.sciencedirect.com/science/article/pii/S1046592825001846
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Author Notes:Alexis Clavier, Santiago Gómez-Evain, Toshinobu Shida, Rubaba R. Abanti, Franziska Hammerstein, Pavel Kielkowski, Mila Leuthold, Anne K. Schütz
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Summary: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 HBx1-120 isoform, comprising the N-terminal 120 residues, is highly expressed. Here, we describe a protocol for the recombinant overexpression and purification of untagged HBx1-120 from bacterial cells. The procedure is compatible with stable isotope labelling in minimal media. Following cell lysis, HBx1-120 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 HBx1-120 for structural and functional in vitro studies.
Item Description:Online verfügbar: 03. November 2025, Artikelversion: 05. November 2025
Gesehen am 13.02.2026
Physical Description:Online Resource
ISSN:1096-0279
DOI:10.1016/j.pep.2025.106842

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