title: Expression and purification of human farnesoid X receptor-ligand binding domain as soluble form using a dual cistronic expression vector.
abstract: In this study, we show the expression and purification of the human recombinant farnesoid X receptor (FXR)- ligand binding domain (LBD) protein in E. coli using a double cistronic vector, pACYCDuet-1, as a soluble form. We describe here the expression and characterization of a biologically active FXR-LBD(248-476). When expressed in the influence of bacterial promoters (P(T7) and P(Tac)) of the single cistronic expression vectors, the human recombinant FXR-LBD(248-476) was found to be totally insoluble. However, by using a double cistronic expression vector, we were able to obtain the human recombinant FXR-LBD(248-476) in a soluble form. To allow for biological activities, we have subcloned into the pACYCDuet-1 vector, expressed in E. coli cells at some optimized conditions, and purified and characterized the human recombinant active FXR-LBD(248-476) proteins using the fluorescence polarization assay. This suggests that the expression of FXR-LBD in a double cistronic vector improves its solubility and probably assists its correct folding for the biologically active form of the proteins. We suggest that this may represent a new approach to high expression of other nuclear receptors and may be useful as well for other classes of heterodimeric protein partners.
authors: Department of Biotechnology, College of Biomedical and Health Science, Konkuk University, Chungju-City, 380-701, Korea.
authors: Hyun
authors: H
authors: Kang
authors: Micheal B
authors: MB
authors: Ye
authors: Young Yil
authors: YY
authors: Bahk
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005796:Genes; D005822:Genetic Vectors; D006801:Humans; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D000099150:Receptor, Farnesoid X-Activated
chemical_list: D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D000099150:Receptor, Farnesoid X-Activated