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Title 

Ligand-binding domain of farnesoid X receptor(FXR) revealed the best sensitivity and activity among FXR variants from a fluorescence-based assay

Authors 

Kyung Hyun ChoJi Young ParkJang Il HanTae Sook Jeong

Publisher 

Springer Verlag (Germany)

Issue Date 

2003

Citation 

Lipids, vol. 38, no. 11, pp. 1149-1156

Keywords 

steroid receptorscell nucleus receptorchenodeoxycholic acidcholesterol 7alpha monooxygenasefarnesoid X receptorhormone receptoroxysterolsteroid receptorDNA bindingexpression vector

Abstract 

The farnesoid X receptor (FXR, NR1H4) has been recognized as an attractive therapeutic target because it is a nuclear hormone receptor that controls the expression level of cholesterol-7α-hydroxylase, which in turn regulates bile acid production and cholesterol excretion. To compare receptor activity between each domain and the full-length protein, human FXR cDNA was cloned from a human liver cDNA library. Three human FXR cDNA, designated FXR20, FXR33, and FXR53 cDNA, were subcloned and ligated into a pET28a expression vector. Each protein was expressed in Escherichia coli (BL21) and purified by nickel-nitrilotriacetic acid column chromatography. Approximately 5 mg of FXR33 (1-182 amino acids deleted from FXR, 37 kDa) and 2 mg of FXR53 (the full-length protein of FXR, 59 kDa) was purified from 1 L of Luria-Bertani culture, achieving at least 90% purity. The coactivator recruitment assay for FXR activation was carried out with the three variants of the FXR protein by using dissociation-enhanced lanthanide fluoroimmunoassay-europium-N1-labeled anti-His antibody. From an optimized assay, a saturated hyperbolic fluorescence signal curve was produced when 250 nM of FXR33 and 100 nM of steroid receptor coactivator-1 peptide, a coactivator of FXR consisting of 26 amino acids, were used with a concentration dependence on chenodeoxycholic acid (from 0 to 200 pM). The ligand-binding domain of FXR (FXR33) was the most suitable protein for studying the activation of FXR with a fluorescence-based assay, because it showed better structural stability than either the full length of FXR (FXR 53) or the DNA-binding domain of FXR (FXR20).

ISSN 

0024-4201

Link 

http://dx.doi.org/10.1007/s11745-003-1173-y

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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