Tropical Journal of Pharmaceutical Research

Original Research Article | OPEN ACCESS

Biological Activities of Recombinant Liver X Receptor β-Ligand Binding Domain Protein in Tetracycline-Inducible expression System

Hyun Kang

Department of Medical Laboratory Science, College of Health Science, Dankook University, Cheonan-si, Chungnam, 330-714, Republic of Korea;

For correspondence:- Email: hkang@dankook.ac.kr Tel:+82415501452

Received: 11 May 2014 Accepted: 9 July 2014 Published: 18 August 2014

Citation: Kang H. Biological Activities of Recombinant Liver X Receptor β-Ligand Binding Domain Protein in Tetracycline-Inducible expression System. Trop J Pharm Res 2014; 13(8):1247-1255 doi: 10.4314/tjpr.v13i8.8

© 2014 The authors.
This is an Open Access article that uses a funding model which does not charge readers or their institutions for access and distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0) and the Budapest Open Access Initiative (http://www.budapestopenaccessinitiative.org/read), which permit unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited..

Abstract

Purpose: To investigate tetracycline-inducible expression system for producing clinically usable, high-quality 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 15 °C when compared to those induced at 37 ºC 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 ^6;-LBD, Tetracycline-inducible expression system, Fluorescence polarization assay