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

Hiroaki Tanabe,Toshiaki Hosaka,Kunio Hirata,Miki Okada-Iwabu,Takeshi Murata,Noboru Ohsawa,Masato Iwabu,Mikako Shirouzu,Mariko Ikeda,So Iwata,Yoshihiro Nakamura,Shigeyuki Yokoyama,Takaho Terada,Motoaki Wakiyama,Masakatsu Hato,Satoshi Ogasawara,Takashi Kadowaki,Yoshiaki Kawano,Toshimasa Yamauchi,Masaki Yamamoto,K Motoyama ,Tomomi Kimura‐Someya ,Yuki Fujii ,Tsuneo Hino

doi:10.1007/s10969-014-9192-z

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.

Highlights

The adiponectin receptors (AdipoR1 and AdipoR2) are membrane proteins with seven transmembrane helices
The AdipoR1D88 and AdipoR2D99 proteins were crystallized with the anti-AdipoR1 monoclonal antibody Fv fragment, by the lipidic mesophase method
For large-scale preparations of the full-length AdipoR1, AdipoR1D88, and AdipoR2D99 proteins, frozen cells were thawed in high osmotic buffer [10 mM HEPES–NaOH buffer containing 1.0 M NaCl, 10 mM MgCl2, 20 mM KCl, and EDTA-free Complete Protease Inhibitor Cocktail (Roche)], and disrupted by Dounce homogenization

Summary

Introduction

The adiponectin receptors (AdipoR1 and AdipoR2) are membrane proteins with seven transmembrane helices. Kadowaki (&) Department of Diabetes and Metabolic Diseases, Graduate School of Medicine, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan e-mail: tyamau-tky@umin.net separated intact AdipoR1D88 did not undergo further aggregation, and was successfully purified to homogeneity by gel filtration chromatography. The AdipoR1D88 and AdipoR2D99 proteins were crystallized with the anti-AdipoR1 monoclonal antibody Fv fragment, by the lipidic mesophase method.

Results

Conclusion