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Expression, purification, crystallization, and preliminary X-ray crystallographic studies of the human adiponectin receptors, AdipoR1 and AdipoR2

DOI: 10.1007/s10969-014-9192-z DOI Help

Authors: Hiroaki Tanabe (RIKEN Systems and Structural Biology Center; The University of Tokyo) , Kanna Motoyama (RIKEN Systems and Structural Biology Center) , Mariko Ikeda (RIKEN Systems and Structural Biology Center; , RIKEN Center for Life Science Technologies) , Motoaki Wakiyama (RIKEN Systems and Structural Biology Center) , Takaho Terada (RIKEN Systems and Structural Biology Center) , Noboru Ohsawa (RIKEN Systems and Structural Biology Center) , Toshiaki Hosaka (RIKEN Center for Life Science Technologies) , Masakatsu Hato (RIKEN Systems and Structural Biology Center) , Yoshifumi Fujii (RIKEN Systems and Structural Biology Center) , Yoshihiro Nakamura (RIKEN Systems and Structural Biology Center) , Satoshi Ogasawara (Kyoto University) , Tomoya Hino (Kyoto University) , Takeshi Murata (Kyoto University) , So Iwata (Kyoto University, Diamond Light Source) , Miki Okada-iwabu (The University of Tokyo) , Masato Iwabu (The University of Tokyo) , Kunio Hirata (RIKEN SPring-8 Center) , Yoshiaki Kawano (RIKEN SPring-8 Center) , Masaki Yamamoto (RIKEN SPring-8 Center) , Tomomi Kimura-someya (RIKEN Systems and Structural Biology Center)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Structural And Functional Genomics

State: Published (Approved)
Published: January 2015

Open Access Open Access

Abstract: The adiponectin receptors (AdipoR1 and AdipoR2) are membrane proteins with seven transmembrane helices. These receptors regulate glucose and fatty acid metabolism, thereby ameliorating type 2 diabetes. The full-length human AdipoR1 and a series of N-terminally truncated mutants of human AdipoR1 and AdipoR2 were expressed in insect cells. In small-scale size exclusion chromatography, the truncated mutants AdipoR1Δ88 (residues 89–375) and AdipoR2Δ99 (residues 100–386) eluted mostly in the intact monodisperse state, while the others eluted primarily as aggregates. However, gel filtration chromatography of the large-scale preparation of the tag-affinity-purified AdipoR1Δ88 revealed the presence of an excessive amount of the aggregated state over the intact state. Since aggregation due to contaminating nucleic acids may have occurred during the sample concentration step, anion-exchange column chromatography was performed immediately after affinity chromatography, to separate the intact AdipoR1Δ88 from the aggregating species. The separated intact AdipoR1Δ88 did not undergo further aggregation, and was successfully purified to homogeneity by gel filtration chromatography. The purified AdipoR1Δ88 and AdipoR2Δ99 proteins were characterized by thermostability assays with 7-diethylamino-3-(4-maleimidophenyl)-4-methyl coumarin, thin layer chromatography of bound lipids, and surface plasmon resonance analysis of ligand binding, demonstrating their structural integrities. The AdipoR1Δ88 and AdipoR2Δ99 proteins were crystallized with the anti-AdipoR1 monoclonal antibody Fv fragment, by the lipidic mesophase method. X-ray diffraction data sets were obtained at resolutions of 2.8 and 2.4 Å, respectively.

Journal Keywords: Membrane protein; Adiponectin receptors AdipoR1 and AdipoR2; Purification; Antibody; Crystallization; Lipidic mesophase

Subject Areas: Biology and Bio-materials


Instruments: I24-Microfocus Macromolecular Crystallography

Other Facilities: SPring-8 SLS

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