Expression, purification and characterization of hepatitis B virus X protein BH3-like motif-linker-Bcl-xL fusion protein for structural studies

Hideki Kusunoki; Toshiyuki Tanaka; Toshiyuki Kohno; Hirokazu Kimura; Kazuo Hosoda; Kaori Wakamatsu; Isao Hamaguchi

doi:10.1016/j.bbrep.2016.12.006

Expression, purification and characterization of hepatitis B virus X protein BH3-like motif-linker-Bcl-x_L fusion protein for structural studies

Biochem Biophys Rep. 2016 Dec 24:9:159-165. doi: 10.1016/j.bbrep.2016.12.006. eCollection 2017 Mar.

Authors

Hideki Kusunoki¹, Toshiyuki Tanaka², Toshiyuki Kohno³, Hirokazu Kimura⁴, Kazuo Hosoda⁵, Kaori Wakamatsu⁵, Isao Hamaguchi¹

Affiliations

¹ Department of Research on Blood and Biological Products, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan.
² Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan.
³ Department of Biochemistry, Kitasato University School of Medicine, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0374, Japan.
⁴ Infectious Disease Surveillance Center, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan.
⁵ Department of Molecular Science, Graduate School of Science and Technology, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan.

Abstract

Hepatitis B virus X protein (HBx) is a multifunctional protein that interacts directly with many host proteins. For example, HBx interacts with anti-apoptotic proteins, Bcl-2 and Bcl-x_L, through its BH3-like motif, which leads to elevated cytosolic calcium levels, efficient viral DNA replication and the induction of apoptosis. To facilitate sample preparation and perform detailed structural characterization of the complex between HBx and Bcl-x_L, we designed and purified a recombinant HBx BH3-like motif-linker-Bcl-x_L fusion protein produced in E. coli. The fusion protein was characterized by size exclusion chromatography, circular dichroism and nuclear magnetic resonance experiments. Our results show that the fusion protein is a monomer in aqueous solution, forms a stable intramolecular complex, and likely retains the native conformation of the complex between Bcl-x_L and the HBx BH3-like motif. Furthermore, the HBx BH3-like motif of the intramolecular complex forms an α-helix. These observations indicate that the fusion protein should facilitate structural studies aimed at understanding the interaction between HBx and Bcl-x_L at the atomic level.

Keywords: BH3, Bcl-2 homology 3; BH3-like motif; Bcl-2, B-cell lymphoma 2; Bcl-xL; Bcl-xL, B-cell lymphoma extra large; Fusion protein; HBV, hepatitis B virus; HBx; HBx, hepatitis B virus X protein; HCC, hepatocellular carcinoma; HSQC, heteronuclear single quantum coherence; Purification; Structural characterization.