Ligand-binding domain of farnesoid X receptor (FXR) had the highest sensitivity and activity among FXR variants in a fluorescence-based assay.
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-7alpha-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 microM). 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).
Cho
Kyung-Hyun
KH
Lipid Metabolism and Atherosclerosis Research Unit, Korea Research Institute of Bioscience and Biotechnology, Yuseong, Daejeon, South Korea, 305-333.
Park
Ji-Young
JY
Han
Jang-Il
JI
Jeong
Tae-Sook
TS
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D005822:Genetic Vectors; D006801:Humans; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D020033:Protein Isoforms; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017385:Sequence Homology; D014157:Transcription Factors; D000099150:Receptor, Farnesoid X-Activated
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D008024:Ligands; D010455:Peptides; D020033:Protein Isoforms; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; D000099150:Receptor, Farnesoid X-Activated