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.