Purification of functional full-length liver X receptor beta produced in Escherichia coli.
Liver X receptor beta (LXRbeta) is a ligand dependent transcription factor that is a member of the nuclear receptor superfamily. LXRbeta and its isoform LXRalpha have recently been recognized as important regulators of lipid homeostasis in vertebrates. N-terminally hexahistidine-tagged rat LXRbeta was expressed in Escherichia coli as a full-length protein and purified in two chromatographic steps, immobilized metal affinity chromatography and gel filtration. From 10g of bacterial cells, 2.5mg of protein was recovered. The purified LXRbeta is functional with respect to ligand-, DNA-, and coactivator-binding. The synthetic ligand T0901317 bound to LXRbeta with high affinity yielding a K(d) of 2.7nM. Specific interaction with DR4 response elements, in the presence of RXR, was demonstrated with electrophoretic mobility shift assay. Furthermore, surface plasmon resonance analysis of LXRbeta binding to coactivator peptides revealed a ligand dependent interaction with the C-terminal nuclear receptor binding site of the coactivator RAP250. The purified LXRbeta constitutes an important tool for further functional and structural studies.
Toresson
Gudrun
G
Department of Biosciences, Novum, Karolinska Institute, Huddinge SE-14157, Sweden. gudrun.toresson@biosci.ki.se
Schuster
Gertrud U
GU
Steffensen
Knut R
KR
Bengtsson
Martin
M
Ljunggren
Jan
J
Dahlman-Wright
Karin
K
Gustafsson
Jan-Ake
JA
D001483:Base Sequence; D015153:Blotting, Western; D017931:DNA Primers; D004268:DNA-Binding Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics; D000071518:Liver X Receptors; D057093:Orphan Nuclear Receptors; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
D017931:DNA Primers; D004268:DNA-Binding Proteins; D000071518:Liver X Receptors; C000605215:Nr1h3 protein, rat; D057093:Orphan Nuclear Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins