Purification and Reconstitution of Human NTCP

Human NTCP proteins were expressed in High Five insect cells (Thermo Fisher Scientific) using the Bac-to-Bac baculovirus expression system (Thermo Fisher Scientific), and cultured cells were disrupted by homogenization. The membrane fraction was isolated and solubilized with 1% dodecylmaltoside (DDM; Anatrace, Maumee, OH). Solubilized proteins were purified with StrepTactin affinity beads (StrepTactin Sepharose; GE Healthcare, Chicago, IL), an anion-exchange column (HiTrap Q HP; GE Healthcare), and a size exclusion column (Superose 6 Increase; GE Healthcare) in Tris-HCl buffer (pH 7.0) containing 0.1 M NaCl, 0.05% DDM, and 0.002% cholesteryl hemisuccinate (Anatrace). Purified proteins were reconstituted into liposomes consisting of egg phosphatidylcholine with the adjuvant lipid A (50 (link), 51 (link)).

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Takemori T., Sugimoto-Ishige A., Nishitsuji H., Futamura Y., Harada M., Kimura-Someya T., Matsumoto T., Honma T., Tanaka M., Yaguchi M., Isono K., Koseki H., Osada H., Miki D., Saito T., Tanaka T., Fukami T., Goto T., Shirouzu M., Shimotohno K, & Chayama K. (2022). Establishment of a Monoclonal Antibody against Human NTCP That Blocks Hepatitis B Virus Infection. Journal of Virology, 96(5), e01686-21.

Publication 2022

High Five insect cells Bac-to-Bac baculovirus expression system StrepTactin Sepharose HiTrap Q HP Superose 6 Increase cholesteryl hemisuccinate

Adjuvant Anion Baculovirus Cells Cholesteryl hemisuccinate Cultured cells Dodecylmaltoside Human ntcp proteins Insect Lipid a Liposomes Membrane Nacl Phosphatidylcholine Proteins Sepharose Tris buffer

Corresponding Organization : National Center for Global Health and Medicine

Other organizations : Fujita Health University, RIKEN Center for Sustainable Resource Science, RIKEN Center for Biosystems Dynamics Research, Wakayama Medical University

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Variable analysis

independent variables

Expression of Human NTCP proteins in High Five insect cells using the Bac-to-Bac baculovirus expression system
Solubilization of membrane fraction with 1% dodecylmaltoside (DDM)
Purification of solubilized proteins using StrepTactin affinity beads, an anion-exchange column, and a size exclusion column
Reconstitution of purified proteins into liposomes consisting of egg phosphatidylcholine with the adjuvant lipid A

dependent variables

Not explicitly mentioned

control variables

Tris-HCl buffer (pH 7.0) containing 0.1 M NaCl, 0.05% DDM, and 0.002% cholesteryl hemisuccinate

controls

Not specified

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