title: Overexpression, refolding, and purification of polyhistidine-tagged human retinoic acid related orphan receptor RORalpha4.
abstract: RORalpha4 is a nuclear receptor activating the transcription of genes that are important for a variety of physiological processes like muscle differentiation, lipid and bone metabolism, cerebellar development, and inflammation. Furthermore, it plays an essential role in maintaining circadian rhythmicity of the core clock in the suprachiasmatic nuclei (SCN). Here, we describe the successful overexpression and purification of human full-length RORalpha4 in Escherichia coli using a T7 expression system. The expressed protein formed inclusion bodies which were solubilized in the presence of 6M guanidinium-HCl and renatured by gradual removal of guanidinium-HCl and addition of l-arginine. The refolded protein was purified by nickel affinity chromatography due to an N-terminal polyhistidine tag which can be cleaved with thrombin subsequently. This method permitted us to obtain up to 20mg of pure and native RORalpha4 protein per liter of E. coli culture. The DNA binding activity of the refolded protein was demonstrated by electrophoretic mobility shift assay (EMSA) using an oligonucleotide comprising the ROR-response element (RORE) motif (A/G)GGTCA. In addition, we developed a new monoclonal antibody to human RORalpha in mice with high sensitivity and specificity.
authors: Institute of Pharmaceutical Chemistry/ZAFES, Johann Wolfgang Goethe University Frankfurt, Max-von-Laue-Strasse 9, D-60438 Frankfurt am Main, Germany.
authors: Adriane
authors: A
authors: Lechtken
authors: Ilse
authors: I
authors: Zündorf
authors: Theo
authors: T
authors: Dingermann
authors: Beate
authors: B
authors: Firla
authors: Dieter
authors: D
authors: Steinhilber