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Ligand-binding domain of farnesoid X receptor (FXR) had the highest sensitivity and activity among FXR variants in a fluorescence-based assay

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Lipids

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 μM). 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 (FXR53) or the DNA-binding domain of FXR (FXR20).

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Abbreviations

apo:

apolipoprotein

bp(s):

base pair(s)

CDCA:

chenodeoxycholic acid

CYP7A1:

cholesterol-7α-hydroxylase

DELFIA:

dissociation-enhanced lanthanide fluoroimmunoassay

6-ECDCA:

6α-ethyl-chenodeoxycholic acid

FXR:

farnesoid X receptor

1-BABP:

ileal-bile acid-binding protein

Ni-NTA:

nickel-nitrilotriacetic acid

PLTP:

phospholipid transfer protein

SHP:

small heterodimer partner

SRC-1:

steroid receptor coactivator-1

TBST:

Tris-HCL+NaCl+EDTA+Tween

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Authors and Affiliations

  1. Lipid metabolism and Atherosclerosis Research Unit, Korea Research Institute of Bioscience and Biotechnology, 52 Eoun-dong, Yuseong, 305-333, Daejeon, South Korea

    Kyung-Hyun Cho, Ji-Young Park, Jang-Il Han & Tae-Sook Jeong

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  1. Kyung-Hyun Cho
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Correspondence to Tae-Sook Jeong.

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Cho, KH., Park, JY., Han, JI. et al. Ligand-binding domain of farnesoid X receptor (FXR) had the highest sensitivity and activity among FXR variants in a fluorescence-based assay. Lipids 38, 1149–1156 (2003). https://doi.org/10.1007/s11745-003-1173-y

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  • DOI: https://doi.org/10.1007/s11745-003-1173-y

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