title: Expression, purification, crystallization, and preliminary X-ray crystallographic studies of the human adiponectin receptors, AdipoR1 and AdipoR2.
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.
authors.affiliation: RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Tanabe
authors.forename: Kanna
authors.initials: K
authors.lastname: Motoyama
authors.forename: Mariko
authors.initials: M
authors.lastname: Ikeda
authors.forename: Motoaki
authors.initials: M
authors.lastname: Wakiyama
authors.forename: Takaho
authors.initials: T
authors.lastname: Terada
authors.forename: Noboru
authors.initials: N
authors.lastname: Ohsawa
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Hosaka
authors.forename: Masakatsu
authors.initials: M
authors.lastname: Hato
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Fujii
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Nakamura
authors.forename: Satoshi
authors.initials: S
authors.lastname: Ogasawara
authors.forename: Tomoya
authors.initials: T
authors.lastname: Hino
authors.forename: Takeshi
authors.initials: T
authors.lastname: Murata
authors.forename: So
authors.initials: S
authors.lastname: Iwata
authors.forename: Miki
authors.initials: M
authors.lastname: Okada-Iwabu
authors.forename: Masato
authors.initials: M
authors.lastname: Iwabu
authors.forename: Kunio
authors.initials: K
authors.lastname: Hirata
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Kawano
authors.forename: Masaki
authors.initials: M
authors.lastname: Yamamoto
authors.forename: Tomomi
authors.initials: T
authors.lastname: Kimura-Someya
authors.forename: Mikako
authors.initials: M
authors.lastname: Shirouzu
authors.forename: Toshimasa
authors.initials: T
authors.lastname: Yamauchi
authors.forename: Takashi
authors.initials: T
authors.lastname: Kadowaki
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D002850:Chromatography, Gel; D003460:Crystallization; D018360:Crystallography, X-Ray; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D050505:Mutant Proteins; D009154:Mutation; D066329:Protein Aggregates; D011485:Protein Binding; D055550:Protein Stability; D054419:Receptors, Adiponectin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D013696:Temperature; D014961:X-Ray Diffraction
chemical_list: C475418:ADIPOR1 protein, human; C475420:ADIPOR2 protein, human; D050505:Mutant Proteins; D066329:Protein Aggregates; D054419:Receptors, Adiponectin