Expression, purification and characterization of hepatitis B virus X protein BH3-like motif-linker-Bcl-xL fusion protein for structural studies
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-xL, through its BH3-like motif, which leads to elevated cytosolic calcium levels, efficient viral DNA replication and...
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| Format: | Article |
| Language: | English |
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Elsevier
2017-03-01
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| Series: | Biochemistry and Biophysics Reports |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S240558081630156X |
| _version_ | 1818417138389483520 |
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| author | Hideki Kusunoki Toshiyuki Tanaka Toshiyuki Kohno Hirokazu Kimura Kazuo Hosoda Kaori Wakamatsu Isao Hamaguchi |
| author_facet | Hideki Kusunoki Toshiyuki Tanaka Toshiyuki Kohno Hirokazu Kimura Kazuo Hosoda Kaori Wakamatsu Isao Hamaguchi |
| author_sort | Hideki Kusunoki |
| collection | DOAJ |
| description | 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-xL, 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-xL, we designed and purified a recombinant HBx BH3-like motif-linker-Bcl-xL 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-xL 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-xL at the atomic level. |
| first_indexed | 2024-12-14T12:02:01Z |
| format | Article |
| id | doaj.art-5bbc149269ac49f686dedf55ae4934cc |
| institution | Directory Open Access Journal |
| issn | 2405-5808 |
| language | English |
| last_indexed | 2024-12-14T12:02:01Z |
| publishDate | 2017-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Biochemistry and Biophysics Reports |
| spelling | doaj.art-5bbc149269ac49f686dedf55ae4934cc2022-12-21T23:01:58ZengElsevierBiochemistry and Biophysics Reports2405-58082017-03-019C15916510.1016/j.bbrep.2016.12.006Expression, purification and characterization of hepatitis B virus X protein BH3-like motif-linker-Bcl-xL fusion protein for structural studiesHideki Kusunoki0Toshiyuki Tanaka1Toshiyuki Kohno2Hirokazu Kimura3Kazuo Hosoda4Kaori Wakamatsu5Isao Hamaguchi6Department of Research on Blood and Biological Products, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, JapanGraduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, JapanDepartment of Biochemistry, Kitasato University School of Medicine, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0374, JapanInfectious Disease Surveillance Center, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, JapanDepartment of Molecular Science, Graduate School of Science and Technology, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, JapanDepartment of Molecular Science, Graduate School of Science and Technology, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, JapanDepartment of Research on Blood and Biological Products, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, JapanHepatitis 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-xL, 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-xL, we designed and purified a recombinant HBx BH3-like motif-linker-Bcl-xL 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-xL 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-xL at the atomic level.http://www.sciencedirect.com/science/article/pii/S240558081630156XBH3-like motifBcl-xLFusion proteinHBxPurificationStructural characterization |
| spellingShingle | Hideki Kusunoki Toshiyuki Tanaka Toshiyuki Kohno Hirokazu Kimura Kazuo Hosoda Kaori Wakamatsu Isao Hamaguchi Expression, purification and characterization of hepatitis B virus X protein BH3-like motif-linker-Bcl-xL fusion protein for structural studies Biochemistry and Biophysics Reports BH3-like motif Bcl-xL Fusion protein HBx Purification Structural characterization |
| title | Expression, purification and characterization of hepatitis B virus X protein BH3-like motif-linker-Bcl-xL fusion protein for structural studies |
| title_full | Expression, purification and characterization of hepatitis B virus X protein BH3-like motif-linker-Bcl-xL fusion protein for structural studies |
| title_fullStr | Expression, purification and characterization of hepatitis B virus X protein BH3-like motif-linker-Bcl-xL fusion protein for structural studies |
| title_full_unstemmed | Expression, purification and characterization of hepatitis B virus X protein BH3-like motif-linker-Bcl-xL fusion protein for structural studies |
| title_short | Expression, purification and characterization of hepatitis B virus X protein BH3-like motif-linker-Bcl-xL fusion protein for structural studies |
| title_sort | expression purification and characterization of hepatitis b virus x protein bh3 like motif linker bcl xl fusion protein for structural studies |
| topic | BH3-like motif Bcl-xL Fusion protein HBx Purification Structural characterization |
| url | http://www.sciencedirect.com/science/article/pii/S240558081630156X |
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