Expression, purification, crystallization, and preliminary X-ray crystallographic studies of the human adiponectin receptors, AdipoR1 and AdipoR2
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- 作者：Hiroaki Tanabe ; Kanna Motoyama ; Mariko Ikeda…
- 关键词：Membrane protein ; Adiponectin receptors AdipoR1 and AdipoR2 ; Purification ; Antibody ; Crystallization ; Lipidic mesophase
- 刊名：Journal of Structural and Functional Genomics
- 出版年：2015
- 出版时间：March 2015
- 年：2015
- 卷：16
- 期：1
- 页码：11-23
- 全文大小：3,712 KB
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- 出版者：Springer Netherlands
- ISSN：1570-0267
文摘
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 89-75) and AdipoR2Δ99 (residues 100-86) 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??, respectively.