Effects of Co-Expression of Liver X Receptor Β-Ligand Binding Domain with Its Partner, Retinoid X Receptor Α-Ligand Binding Do

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

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

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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. (C) The double cistronic expression construct in polarization assays in the assay buffer. Varied concentrations of the pACYCDuet-1 expression vector. LXR β-LBD was fused to (His)6 LXR β-LBD/RXR α-LBD in the presence or absence of T-0901317 at its N-terminal and subcloned into the 1st multiple cloning site, were incubated at room temperature with a europirim-labeled whereas RXR α-LBD was not fused to any tag and subcloned into the peptide. The polarization degrees (FP) were measured with an 2nd site. excitation wavelength of 615 nm and an emission wavelength of 665 nm. The apparent dissociation constant values were determined by the binding curves derived from a non-linear least-squares fit an optical density of 0.8-1.0 (detected at 600 nm) using 0.4 mM of the data for a sample 1:1 interaction. isopropyl β-D-thiogalactopyranoside (IPTG) and grown for a further 5-6 h before harvesting by centrifugation. The collected Western Blot Analysis and Antibodies cell pellets were resuspended in buffer A containing 50 mM Equal amounts of protein were loaded and resolved by 12% Tris-HCl, pH 8.0, 100 mM KCl, and 1 mM EDTA for purifying SDS-PAGE. Resolved proteins were transferred to an NC glutathione S-transferase (GST) fusion protein or in buffer B membrane (S&S, Dassel, Germany), blocked with 5% non-fat dry containing 20 mM HEPES, pH 8.0, 100 mM HCl, and 20 mM milk in TBS, and probed with specific antibodies against (His)6- imidazole for purifying the (His)6-tagged proteins. The cells were tag, GST, RXR α, and LXR β (Cell Signaling Technology, Beverly, lysed by ultrasonication in the above-mentioned buffers containing MA, USA), followed by the secondary antibodies coupled to Protease Inhibitor Cocktail (Roche Diagnostic, Manheim, horseradish peroxidase (Bio-Rad, Hercules, CA, USA). The Germany). After lysis by ultrasonication, the lysate was cleared immunoreactive proteins on the membrane were detected by by centrifugation and analyzed immediately or stored at -80°C. chemiluminescence using the Western Blotting Detection Reagent For purification, the lysates were loaded at 4oC onto a Ni+2- Kit (AbSignal, AbClon Co., Seoul, Korea). nitrilotriacetate acid (NTA) agarose column (Probonds Resin, Invitrogen). The agarose columns were washed with either buffer Results A or buffer B. The proteins were eluted from the column in the same buffer containing the appropriate concentration of imidazole. Expression of LXR β-LBD Proteins from PT7 and PTac The fractionation was performed with a Source 15Q anion- Promoters as Single Cistronic Plasmid Construction exchange column (GE Healthcare, Piscataway, NJ, USA, column Nr1b2 (LXR β-LBD) was cloned into the pET-15b expression volume (CV) ¼ 3.9 ml) with an NaCl gradient. Fifteen milliliters of vector to produce LBD tagged at the N-terminus with six the desalted lysate (15 mg of total protein) was loaded and eluted histidine residues, and also into the pGEX-4T-1 vector so as with three-step linear gradients (25–200 mM NaCl for 1.5 CV, 200– 300mM NaCl for 4 CV, and 300–700 mM for 2 CV) to evenly to express a GST-tagged protein. The clones were used to distribute the proteins into separate fractions. Fifteen 1.5 ml transform E. coli BL21 (DE3) pro for an initial expression fractions were collected and used immediately or stored at -80oC. test. E. coli cells, induced to express recombinant protein by

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Fig. 2. Expression of recombinant LXR β-LBD using pET-15b expression vector. Fig. 3. SDS-PAGE analysis of recombinant fusion protein

The LXR β-LBD(205-461) gene was inserted into pET-15b containing a expression (LXR β-LBD(205-461)) using a double cistronic hexahistidine sequence to the construct. Soluble (S) and insoluble (P) expression system with RXR α-LBD(225-462). proteins were separated on an SDS-PAGE gel, subjected to immunoblot Soluble (S) and insoluble (P) recombinant LXR β-LBD(205-461) fusion analysis with anti-(His)6 antibody (A), and stained with Coomassie proteins induced or uninduced at either 20ºC or 30ºC and resolved by Brilliant Blue R-250 (B). The arrow indicates the expressed proteins. SDS-PAGE and stained with Coomassie Blue R-250. The arrow

indicates the position of the recombinant LXR β-LBD(205-461) fusion protein with its binding partner, RXR α-LBD . Lane M represents IPTG, formed both insoluble particulate and soluble (225-462) the calibrated molecular weight markers. fractions. As shown in the right panels of Figs. 1A and 1B and Fig. 2B, LXR β-LBD(205-461) was found to be insoluble at a high level using both single expression constructs. The RXR α-LBD(225-462), its expression partner in the so-called insoluble LXR β-LBD(205-461) could be detected by anti-(His)6 pACYCDuet/HN-LXR β-LBD/RXR α-LBD vector. The antibodies by western blot analysis (Fig. 2A). In addition, double cistronic vectors gave high yields of both soluble the expressed RXR α-LBD could not be pulled down by proteins (Fig. 3). The E. coli BL21 (DE3) cells transformed GST LXR β-LBD on agarose beads (data not shown). These with the pACYCDuet/HN-LXR β-LBD/RXR α-LBD vector results demonstrate that the single citronic pET and pGEX (Fig. 1C) were grown at 37°C and induced with 0.4 mM vectors driven by T7 and Tac promoters failed to support IPTG at either 20°C or 30°C for 5-6h. The cells were the correct folding of the proteins under our experimental harvested and lysed by ultrasonication, and the soluble (S) conditions. and insoluble (P) fractions were collected and analyzed by SDS-PAGE (Fig. 3). Although the two partners (LXR β-LBD Double Cistronic Plasmid Construction, Expression, and and RXR α-LBD) were expressed primarily as insoluble Purification proteins at both temperatures, the recombinant proteins Several lines of evidence suggest that the heterodimeric were detected as soluble proteins (Fig. 3). partners communicate with each other, presumably through The LXR β-LBD was found to be soluble and expressed at their dimer interface and/or their associated co-factors [17]. relatively high levels by co-expressing its binding partner, Thus, I used a commercially available double cistronic RXR α-LBD, using the double cistronic expression vector vector, pACYCDuet-1, that directs expression of the (Figs. 1C and 3). The transformed E. coli cells were harvested heterodimer in a single cell. LXR β-LBD(205-461) was cloned after induction with 0.4 mM IPTG at 30ºC. The bacterial into the pACYCDuet-1 expression vector, which is designed lysate was obtained using an ultrasonicator, and, after to co-express two target genes (Fig. 1C). The vector contains clarification by centrifugation, was loaded onto a Ni+2-NTA two multiple cloning sites, each of which is driven by a T7 agarose column. The column resin was then washed until promoter (PT7) and preceded by a lac operator and ribosome no protein was found in the eluate. The bound proteins binding site (Rbs). In the cistronic construct, LXR β-LBD(205-461) were eluted stepwise using 40, 100, and 300 mM of tagged with (His)6 at its N-terminus is placed upstream of imidazole, purified, resolved by SDS-PAGE, and visualized

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Fig. 4. Analysis of heterodimer formation for the recombinant LXR β-LBD fusion protein with its binding partner, RXR α- LBD, using a double cistronic expression vector. Cell lysates from the induced [IPTG (+)] or uninduced [IPTG (-)] E. coli cells at either 20 ºC or 30 ºC were incubated with either anti-(His)6 antibody (A) or anti-LXR antibody (B) and protein A agarose. The affinity-purified proteins were resolved by SDS-PAGE and subjected to immunoblot analysis with antibodies against the RXR α protein.

with Coomassie Brilliant Blue R-250. In this way, the highly pure (at least 95%) heterodimers of LXR β-LBD and RXR α-LBD were obtained (Fig. 5). To decrease the complexity of the E. coli protein for target identification, the soluble fraction of an E. coli lysate was fractionated by anion- exchange chromatography. The salt gradient was chosen after multiple trials to maximize the distribution of proteins into different fractions. The chromatogram (absorbance at 280 nm) and the salt profile (conductivity of the elution buffer) are shown in Fig. 5A. The eluant from 3 to 20 ml was separated into fifteen 1.5 ml fractions. Proteins that Fig. 5. Purification of the recombinant protein using a double were eluted at the washing step were not used in this cistronic expression vector, pACYCDuet-1. study. SDS-PAGE of the fractions showed that proteins in (A) An E. coli lysate was fractionated by using anion-exchange chromatography. The chromatogram shows the elution monitored by the cell lysate were evenly fractionated (Fig. 5B). Most absorbance at 280 nm (A280; solid line) and the conductivity (dashed fractions contained 30 to 50 easily identifiable protein line). The location of each fraction is indicated by a vertical tick on the bands on the SDS-PAGE gels. x-axis. (B) The protein contents of the fractions were checked by SDS- PAGE. The molecular weight markers (M) and the total lysate (L) Analysis of Ligand Binding were loaded as references. In this study, I used T-0901317 as an agonist to evaluate the biological activity of the recombinant LXR β-LBD/RXR and an emission wavelength of 665 nm. Fig. 6 shows a α-LBD heterodimer. typical dose-response curve obtained by using increasing The effects of T0901317 on the interaction of the co- concentrations of the purified recombinant LXR β-LBD/ activator peptide (SRC-1a, CPSSHSSLTERHKILHRLLQEGSPS) RXR α-LBD and measuring FI as an index of fluorescent and the acceptor APC with the recombinant LXR β-LBD/ peptide binding to LBD. This type of experiment was RXR γ-LBD protein were determined by fluorescence repeated a number of times during the course of this study, polarization assays. Varied concentrations of the purified allowing us to calculate an ED50 of 719 nM. Because our recombinant LXR β-LBD/RXR α-LBD in the presence or assays were performed using only the purified recombinant absence of T-0901317 were incubated at room temperature LXR β-LBD/RXR α-LBD protein, it is likely that the true with a europirim-labeled peptide. The polarization degrees value could somewhat lower. Thus, a protein expression were measured with an excitation wavelength of 615 nm system using the dual cistronic pACYCDuet-1 vector with

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doubling time (rapidity), and inexpensiveness. In fact, preparations to enrich a specific protein are rarely easily obtained from their natural host cells. Hence, recombinant protein production is frequently the sole applicable procedure. With the advent of the post-genomic era has come the need to express in E. coli a growing number of genes originating from different organisms. Recombinant fusion partners and mutant strains have advanced the possibilities with E. coli. To explore the interaction between LXR β-LBD and RXR

α-LBD, the (His)6-tagged LXR β-LBD proteins (and any interacting proteins) were recovered by immunoprecipitation

using antibodies against LXR and (His)6, and the LXR β- LBD immunoprecipitate was subjected to SDS-PAGE and analyzed for the presence of RXR α-LBD. As shown in Fig. 6. Ligand-binding studies of the purified recombinant Figs. 4A and 4B, the recombinant RXR α-LBD protein was protein using a fluorescence polarization assay. observed in both the immunoprecipitates collected with To detect direct binding of probes to synthesized and purified anti-(His) and anti-LXR antibodies. This co-immunoprecipiation heterodimers of LXR β-LBD and RXR α-LBD by the co-activator 6 approach has the advantage of being able to identify the peptide (SRC-1a, CPSSHSSLTERHKILHRLLQEGSPS; Cosmo Genetech Co., Seoul, Korea) and the acceptor APC, a fluorescence polarization direct interaction with LXR β-LBD that is relevant in this assay was performed as described in Materials and Methods. Each biological system. In addition, this dimer was found to be datum point was measured in triplicate. The polarization degree (FP) stable under other conditions (data not shown). From these was plotted against the recombinant protein concentration, and non- experiments, I have demonstrated that the expression of linear regression analysis was performed to determine the ED . 50 LXR β-LBD using the single cistronic expression vectors are in some ways defective for proper folding and biological IPTG induction was found to be suitable for producing activities. However, the bacterially produced heterodimer soluble heterodimers consisting of LXR β-LBD(205-461) and using the double cistronic expression vector is soluble and

RXR α-LBD(225-462) and this should be helpful for future the two dimmers are not easily dissociated. It is clear that biological and structural analyses. co-expression increases the solubility of LXR β-LBD as well as ensuring its proper folding. This phenomenon might be Discussion due to the masking of any hydrophobic patches that can cause LXR β-LBD to form aggregates [22]. High expression of functional proteins in E. coli, especially Our method was used to produce a soluble recombiant those from eukaryotic origin, has often been challenging as protein by using a double cistronic E. coli expression the proteins tend to aggregate in inclusion bodies within system. Because the soluble fraction can be directly applied the bacteria. Recently, the three-dimensional structure of to Ni-NTA columns for purification, there are advantages. LXR β-LBDs was determined, although expression of LXR In addition, use of the pACYCDuet-1 vector further β-LBD in E. coli yielded predominantly insoluble proteins simplifies downstream purification processes, including [7, 24, 27]. The soluble fraction was unstable and could not cleavage and elution. Thus, our approach may be helpful be suitably concentrated for protein crystallization. To obtain for increasing the solubility and yield of other proteins of the active protein, the aggregated protein was solubilized interest. with detergent or denaturing agents and re-folded to The actions of lipophilic hormones, including steroids, recover the protein activity. However, frequently re-folded retinoids, thyroid hormone, and vitamin D3, are mediated proteins may not recover their biological activity. Despite through a conserved superfamily of nuclear receptor some limitations, E. coli remains the most efficient and proteins that function as ligand-regulated, DNA-binding widely used host for recombinant protein production [23]. transcriptional activators [16]. LXRs were initially described The main reasons for using E. coli as a host for expressing as orphan receptors; later, monooxygenated cholesterol recombinant proteins include its well-defined genetics, derivatives, oxysterols, were identified as their natural high transformation efficiency, simple cultivation, short ligands [8, 9]. Recently, two nonsteroid synthetic LXR

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