期刊
ANALYTICAL BIOCHEMISTRY
卷 384, 期 2, 页码 279-287出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2008.10.003
关键词
Dioxin; PCB; Ligand binding; Aryl hydrocarbon receptor; Species difference
资金
- UK Food Standards Agencyx [T01034]
Recombinant expression of the aryl hydrocarbon receptor (AhR) yields small amounts of ligand-binding-competent AhR. Therefore. Spodoptera frugiperda (Sf9) cells and baculovirus have been evaluated for high-level and functional expression of AhR. Rat and human AhR were expressed as soluble protein in significant amounts. Expression of ligand-binding-competent AhR was sensitive to the protein concentration of Sf9 extract, and coexpression of the chaperone p23 failed to affect the yield Of functional ligand-binding AhR. The expression system yielded high levels of functional protein. with the ligand-binding capacity (B-max) typically 20-fold higher than that obtained with rat liver cytosol. Quantitative estimates of the ligand-binding affinity Of human and rat AhR were obtained; the K-d for recombinant rat AhR was indistinguishable from that of native rat AhR, thereby validating the expression system as a faithful model for native AhR. The human AhR bound TCDD with significantly lower affinity than the rat AhR. These findings demonstrate high-level expression of ligand-binding-competent AhR, and sufficient AhR for quantitative analysis of ligand binding. (C) 2008 Elsevier Inc. All rights reserved.
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