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

IF 1.8 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification Pub Date : 2026-02-01 Epub Date: 2025-11-03 DOI:10.1016/j.pep.2025.106842

Alexis Clavier , Santiago Gómez-Evain , Toshinobu Shida , Rubaba R. Abanti , Franziska Hammerstein , Pavel Kielkowski , Mila Leuthold , Anne K. Schütz

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引用次数: 0

Abstract

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.

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人乙型肝炎病毒HBx蛋白癌症相关亚型的表达、纯化和功能验证

人类乙型肝炎病毒（HBV）引起乙型肝炎，这是一种急性或慢性肝脏感染。HBV编码四种蛋白，其中X蛋白（HBx）在病毒复制中起关键作用。在慢性HBV感染期间，病毒DNA被整合到宿主基因组中，包含n端120残基的HBx1-120亚型被高度表达。在这里，我们描述了一种从细菌细胞中重组过表达和纯化未标记HBx1-120的方案。该方法与稳定同位素标记在最小介质中兼容。细胞裂解后，从包涵体（IBs）中回收HBx1-120，在尿素中溶解，并通过离子交换（IEX）和尺寸排除色谱（SEC）纯化。纯化后的蛋白被广泛地表征，包括质谱和核磁共振（NMR）光谱。功能通过与已知的相互作用伙伴Spindlin1的下拉试验确认。该方案为获得用于体外结构和功能研究的未标记HBx1-120提供了一个强大的框架。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Protein expression and purification 生物-生化研究方法

CiteScore

3.70

自引率

6.20%

发文量

120

审稿时长

32 days

期刊介绍： Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.