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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• 作者：Kyung-Hyun Cho (1)
  Ji-Young Park (1)
  Jang-Il Han (1)
  Tae-Sook Jeong (1) tsjeong@kribb.re.kr
• 刊名：Lipids
• 出版时间：November 2003
• 出版年：2003
• 期刊代码：pc361_0024-4201
• 类别：ch
• 卷：38
• 期：11
• 页码：1149-1156
• 数据来源：sp

摘要

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).