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Published:
Sep 9, 2014DOI:
10.4314/tjpr.v13i8.8Keywords:
Article Details
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Main Article Content
Biological Activities of Recombinant Liver X Receptor â- Ligand Binding Domain Protein in Tetracycline-Inducible Expression System
Abstract
Purpose: To investigate tetracycline-inducible expression system for producing clinically usable, highquality liver X receptor ligand-binding domain recombinant protein
Methods: In this study, we have expressed and purified the recombinant liver X receptor β-ligand binding domain proteins in E. coli using a tetracycline inducible system. To allow for biological activities, we subcloned into pPROTet.E HN vector, expressed in E. coli cells under optimized conditions, purified and characterized the recombinant liver X receptor β-ligand-binding domain proteins using fluorescence polarization assay.
Results: The use of pPROTet.E HN vector simplified downstream purification processes, including cleavage and elution thereby increasing the solubility and yield of the protein of interest. There was a 2.3-fold increase in the efficiency of recombinant LXR β-ligand binding domain (LBD) production by optimizing the expression temperature to 15oC when compared to those induced at 37oC during the induction procedures. A typical dose-response curve obtained using increasing concentrations of the
purified recombinant LXR β-LBD (197-461) and measuring fluorescence intensity (FI) as an index of fluorescent peptide binding to LBD showed 50 % effective dose (ED50) value of 533 nM. The recombinant LXR β-LBDs were substantially soluble and should be useful for future biological,
biophysical and structural analyses of nuclear receptor complexes. 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.
Conclusion: These findings indicate that recombinant LXR β-LBD protein is a promising target for the development of molecular ligands with improved therapeutic windows.
Keywords: Nuclear receptor, Recombinant LXR β-LBD, Tetracycline-inducible expression system, Fluorescence polarization assay
