Fan, MingQi, Bell, Alex R., Bell, David Robert, Clode, Sally, Fernandes, Alwyn, Foster, Paul M.D., Fry, Jeffery R., Jiang, tao, Loizou, George, MacNicoll, Alan, Rose, Martin, Miller, Brian G., Shaikh-Omar, Osama, Tran, Lang and White, Shaun
(2009)
Recombinant expression of Aryl Hydrocarbon Receptor
for quantitative ligand-binding analysis.
Analytical Biochemistry, 384
.
pp. 279-287.
ISSN 0003-2697
Full text not available from this repository.
Abstract
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 co-expression 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 (Bmax) 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 Kd 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.
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