Effects of Co-Expression of Liver X Receptor Β-Ligand Binding Domain with Its Partner, Retinoid X Receptor Α-Ligand Binding Do
Total Page：16
File Type：pdf, Size：1020Kb
J. Microbiol. Biotechnol. (2015), 25(2), 247–254 http://dx.doi.org/10.4014/jmb.1406.06013 Research Article Review jmb Effects of Co-Expression of Liver X Receptor β-Ligand Binding Domain with its Partner, Retinoid X Receptor α-Ligand Binding Domain, on their Solubility and Biological Activity in Escherichia coli S Hyun Kang* Department of Medical Laboratory Science, College of Health Science, Dankook University, Cheonan-si 330-714, Republic of Korea Received: June 5, 2014 Revised: August 18, 2014 In this presentation, I describe the expression and purification of the recombinant liver X Accepted: September 15, 2014 receptor β-ligand binding domain proteins in E. coli using a commercially available double First published online cistronic vector, pACYCDuet-1, to express the receptor heterodimer in a single cell as the September 15, 2014 soluble form. I describe here the expression and characterization of a biologically active *Corresponding author heterodimer composed of the liver X receptor β-ligand binding domain and retinoid X Phone: +82-41-550-1452; receptor α-ligand binding domain. Although many of these proteins were previously seen to Fax: +82-41-559-7934; E-mail: [email protected] be produced in E. coli as insoluble aggregates or “inclusion bodies”, I show here that as a form of heterodimer they can be made in soluble forms that are biologically active. This suggests S upplementary data for this that co-expression of the liver X receptor β-ligand binding domain with its binding partner paper are available on-line only at improves the solubility of the complex and probably assists in their correct folding, thereby http://jmb.or.kr. functioning as a type of molecular chaperone. pISSN 1017-7825, eISSN 1738-8872 Copyright© 2015 by Keywords: Nuclear receptor, recombinant LXR β-LBD, RXR α-LBD, dual expression vector, The Korean Society for Microbiology E. coli expression system and Biotechnology Introduction ligand binding and dimerization domain with ligand- dependent AF-2 region [2]. Liver X receptor (LXR, consisting Members of the nuclear receptor superfamily including of two members, LXR α and LXR β, are encoded by the receptors for retinoic acid, vitamin D, thyroid hormone, Nr1b3 and Nr1b2, respectively) is an orphan receptor that and steroid hormones are hormone-activated transcription heterodimerizes with RXR, binds to a specific response factors that have important roles in many physiological element called the LXRE, and has the potential to modulate processes [12, 13]. The hormone nuclear receptor superfamily physiological responses [2]. Both LXRs are widely includes different proteins responsible for the direct link expressed in multiple tissues and cell types, but LXR α is between transcriptional regulation and physiological most highly expressed in the liver and intestine [28]. responses. A subgroup of receptors within the superfamily, The natural ligands for the LXRs consist of a select group including the retinoic acid receptor (RAR), thyroid hormone of oxysterols derived from tissue-specific cholesterol receptor (TR), peroxisome proliferator activated receptor metabolism in the liver, brain, and gonads [21]. Although (PPAR), and many orphan receptors, bind to specific DNA their presumed natural ligand is not cholesterol itself, sequences known as hormone response elements as recent studies suggest that the LXRs serve as one of the heterodimers with the retinoid X receptor (RXR) and share body’s key sensors of dietary cholesterol and therefore a conserved structural and functional framework [12, 14]. The regulate essential pathways in cholesterol homeostasis. nuclear receptors have a modular structure composed of Activated in response to increased levels of intracellular three independent conserved domains; the N-terminal AF-1, cholesterol, LXRs act to induce specific gene expression the highly conserved DNA binding domain, and finally the programs to control various aspects of cholesterol Feburary 2015 ⎪ Vol. 25⎪ No. 2 248 Hyun Kang homeostasis [9, 21]. In addition, LXR signaling negatively receptor heterodimer in a single cell. I describe here the regulates inflammation by repressing the expression of expression and characterization of the LXR β-LBD and its genes involved in the inflammatory response [10, 19]. heterodimeric partner, RXR α-LBD. When expressed alone, The three-dimensional structure of the LXR β-LBDs, in the LXR β-LBD was found to be largely insoluble and the complex with the synthetic agonist T-0901317, has been formation of heterodimeric complexes was not sufficient. determined [7, 24, 27, 29]. Analysis of the structure provides However, by using a double cistronic expression system, I evidence that the heterodimeric partners communicate was able to obtain high levels of soluble, tightly associated with each other, presumably through their dimer interface heterodimers. This suggests that co-expression of LXR β- and also potentially through their associated co-factors [6]. LBD with its binding partner RXR α-LBD improves the Physiologically, activation of LXRs drives the expression of solubility of the complex and probably assists in their genes involved in reverse cholesterol transport, biliary correct folding, thereby functioning as a type of molecular excretion, and intestinal absorption [21], and could lead to chaperone. an increase in HDL particle numbers. In mice, treatment with synthetic agonists results in increased HDL levels and Materials and Methods decreased atherosclerotic lesions [11, 25]. Thus, LXRs may be promising therapeutic targets for treating dyslipidemia Preparation of Expression Constructs and atherosclerosis. LXR β is likely to be a good target even For the expression of (His)6-tagged LXR β-LBD(205-461) and RXR though it is ubiquitously expressed; for example, LXR β α-LBD(225-462), cDNA fragments encoding the human LXR β-LBD agonists would be useful in non-liver tissues such as and RXR α-LBD were obtained by RT-PCR and subcloned into pET-15b (driven by the P promoter; Novagen, Darmstadt, macrophages and adipose tissues. T7 Germany), pGEX-4T-1 (driven by the P promoter; GE However, the development of LXR ligands as therapeutic Tac Healthcare, Piscataway, NJ, USA), and pACYCDuet-1 (driven by agents has been hampered by the increase in hepatic the PT7 promoter; Novagen) vectors (Fig. 1). For the pACYCDuet-1 triglyceride production by the dual LXR agonists [15, 26]. st vector, (His)6-tagged LXR β-LBD(205-461) was inserted into the 1 LXR α is the dominant subtype in the liver, intestine, kidney, nd MCS at the BamHI/HindIII site and RXR α-LBD(225-462) into the 2 spleen, and adipose tissue, whereas LXR β is ubiquitously MCS at the EcoRV/XhoI site. All constructs were confirmed by expressed at very low levels. In fact, LXR α leads to DNA sequencing. The oligonucleotide primers used for PCR-based stimulation of fatty acid synthesis pathways and increased cloning are shown in Table 1. All molecular biology techniques triglyceride levels. Therefore, an LXR β-selective agonist for constructing the expression plasmids were performed using might retain efficacy without deleteriously increasing hepatic standard procedures. lipogenesis [20]. Hence, the identification of LXR β-selective agonists may be of significant therapeutic value. Protein Expression and Purification Our aim was to express and purify the recombinant LXR The expression constructs for LXR β-LBD in pET-15b, pGEX4T-1, and pACYCDuet-1 were used to transform BL21 (DE3) Pro E. coli β-LBD proteins in E. coli using a commercially available (Invitrogen, Carlsbad, CA, USA). Cultures were grown at 37oC in double cistronic vector, pACYCDuet-1, to express the LB medium containing antibiotics. These cultures were induced at Table 1. Primer sequences for the RT-PCR of LXR β-LBD(205-461) and RXR α-LBD(225-462) genes. Name of primer Sequences Sense S-LXR β-LBD (BamHI) for pET-15b 5’-CGCGGATCCGGAGGCAGGCAGCCAGGGCTC-3’β S-LXR β-LBD (BamHI) for pGEX 4T-1 5’-CGTGGATCCGAGGCAGGCAGCCAGGGCTC-3’β S-LXR β-LBD (BamHI) for pACYCDuet-1 5’-CCAGGATCCGGAGGCAGGCAGCCAGGGCTC-3’β S-RXR α-LBD (EcoRV) for pACYCDuet-1 5’-TTGGATATCGTGGGACAAGTTCAGTGAACTC-3’β Common antisense AS- LXR β-LBD (BamHI) for pET-15b 5’GGCGGATCCTCACTCGTGGACGTCCCAG-3’β AS-LXR β-LBD (XhoI) for pGEX 4T-1 5’CCGCTCGAGTCACTCGTGGACGTCCCAG-3’β AS-LXR β-LBD (HindIII) for pACYCDuet-1 5’-CGCAAGCTTTCACTCGTGGACGTCCCAG-3’ β AS-RXR α-LBD (XhoI) for pACYCDuet-1 5’-AGACTCGAGTTCACGGGGAGTGGGTGGCCG-3’ Underlined sequences represent the restriction enzyme recognition sites. J. Microbiol. Biotechnol. Effects of Co-Expression of Liver X Receptor β-Ligand Binding Domain Proteins 249 Chromatography was performed with the AKTA FPLC system (GE Healthcare; Piscataway, NJ, USA). Solubility Analysis E. coli cell pellets were resuspended in 50 mM Tris-HCl, pH 8.0, 100 mM KCl, 1 mM EDTA, and Protease Inhibitor Cocktail, ultrasonicated, and then centrifuged at 3,000 ×g for 20min to remove intact cells and cell debris. The supernatants were then further centrifuged at 10,000 ×g for 20 min to precipitate the insoluble proteins. Soluble proteins were purified from the supernatant. Ligand Binding Using Fluorescence Polarization Assays Fluorescence polarization assays were performed using a Victor Fig. 1. Schematic diagram of the expression constructs. apparatus (Perkin-Elmer Life and Analysis Sciences, Boston, MA, (A and B) The single cistronic expression constructs in pET-15b and USA) according to the method described by Dagher et al. [5] and pGEX-4T-1, and western blot analysis for detecting the recombinant Lévy-Bimbot et al. [18]. Assays were conducted in 96-well black LXR β-LBD proteins using anti-(His)6 and anti-GST antibodies. Tac polystyrene plates. The effects of T-0901317 on the interaction of and T7 promoters (PT7 and PTac) are indicated by arrows. Repressor the co-activator peptide (SRC-1a, CPSSHSSLTERHKILHRLLQEG- binding sites are represented by open boxes and E. coli ribosome SPS; Cosmo Genetech Co., Seoul, Korea) and the acceptor APC binding sites by open circles. S: soluble fraction; P: insoluble with LXR β-LBD/RXR α-LBD were determined by fluorescence particulate fraction.