Soluble Expression and Purification of Receptor Activator of Nuclear Factor-Kappa B Ligand Using Escherichia coli

Abstract

Receptor activator of nuclear factor-kappa B ligand (RANKL) is a critical factor in osteoclastogenesis. It makes osteoclasts differentiate and multinucleate in bone remodeling. In the present study, RANKL was expressed as a soluble maltose binding protein (MBP)-fusion protein using the Escherichia coli maltose binding domain tag system (pMAL) expression vector system. The host cell E. coli DH5α was cultured and induced by isopropyl β-_D-1-thiogalactopyranoside for rRANKL expression. Cells were disrupted by sonication to collect soluble MBP-fused rRANKL. The MBP-fusion rRANKL was purified with MBP Trap affinity chromatography and treated with Tobacco Etch Virus nuclear inclusion endopeptidase (TEV protease) to remove the MBP fusion protein. Dialysis was then carried out to remove binding maltose from the cleaved rRANKL solution. The cleaved rRANKL was purified with a second MBP Trap affinity chromatography to separate unsevered MBP-fusion rRANKL and cleaved MBP fusion protein. The purified rRANKL was shown to have biological activity by performing in vitro cell tests. In conclusion, biologically active rRANKL was successfully purified by a simple two-step chromatography purification process with one column.

Introduction

Bone is a rigid organ that consists of mineral components, collagen matrix, and cells such as osteocytes, osteoclasts, osteoblasts, and bone cells. Bone has various functions. It supports and protects the vital organs of the body, produces white and red blood cells, and stores calcium and minerals. Bone remodeling occurs through bone resorption by the proteolytic action and acidification of the osteoclasts and through bone formation by bone matrix secreted by the osteoblasts. The bone maintains homeostasis by its balanced activity. If the balance of bone-related cell activation is broken, bone resorption is increased by osteoclasts, and bone diseases, such as osteoporosis, can occur [4,5]. Osteoporosis is a bone disease characterized by decreasing bone mass. It is induced by many causes, such as the sharp decrease in estrogen in postmenopausal women, aging, dose formulation of corticoid, smoking, alcohol intake, and so on [6,12,13].

Osteoclasts are only present in the endosteum of bone. They are differentiated from hematopoietic progenitor cells of the mononuclear-phagocytic system by stromal cells. Several growth regulatory factors, hormones, and cytokines influence osteoclast differentiation. Among the cytokines, receptor activator of nuclear factor kappa-B ligand (RANKL) is the main cytokine [10,15]. RANKL is a homotrimeric transmembrane protein of the tumor necrosis factor (TNF) family. It is also called osteoclast differentiation factor (ODF), osteoprotegrin ligand (OPGL), and TNFrelated activation-induced cytokine (TRANCE). It is a surface-bound molecule found on osteoblasts. RANKL expression is stimulated in osteoblast/stromal cells by many factors that are known to stimulate osteoclast formation and activity. RANKL plays a role in bone restoration via the activation and differentiation of osteoclasts through binding to the receptor activator of nuclear factor kappa-B (RANK) on the osteoclast surface [1,16]. On the other hand, osteoprotegerin (OPG), an inhibitor of RANKL that is secreted by osteoblast or stromal cells, inhibits the binding between RANKL and RANK [2,11]. RANKL is the cause of many bone diseases. RANKL plays other roles in different cells. It affects lactation in mammalian epithelial cells [3], and acquired immunity in dendritic cells, and is the cause of osteosarcoma in prostate cancer and breast cancer cells [9].

Many studies to screen materials that may prevent osteoporosis have been conducted with osteoclasts and osteoblasts. In the study of antagonists against osteoclast differentiation, RANKL is the pivotal cytokine. It is therefore necessary to express rRANKL and to purify it [14]. The purpose of this study was to express soluble rRANKL in E. coli and to develop the purification process in order to get high-purity rRANKL by a simple process with only one chromatography column. After purification, the biological activity of the purified rRANKL was tested.

 

Materials and Methods

Construction of the rRANKL Expression Vector and Cell Culture

The cDNA encoding Lys 158~Asp 316 amino acids of murine RANKL was amplified through the PCR technique. The restriction enzyme sites for NdeI and XhoI were inserted in the forward and reverse primers. The nucleotide sequences are listed: forward primer, 5’-CCAGCATATGAAGCCTGAGGCCCAGCCATTTGCACAC-3’; and reverse primer, 5’-TACGCTCGAGTCAGTCTATGTCCTGAACTTTGAAAGCCCC-3’. The vector system was modified pMAL-c2x, which was provided by Prof. Sung-Gun Kim of Youngdong University, and the constructed expression vector was named pMAL-RANKL. It was transformed into the E. coli DH5α strain. E. coli DH5α containing pMAL-RANKL was cultured in 500 ml of LB medium. When the transformed cells were cultured, RANKL was expressed as a soluble MBP fusion form. Cells were grown to OD600 of 0.4~0.6 at 37℃and then isopropyl-β-D-thiogalactopyranoside (IPTG) was added at a final concentration of 1 mM in order to induce MBP-fusion rRANKL protein expression. After induction, cells were cultured for 21 h at 25℃.

The First Chromatography for rRANKL Purification with MBP Affinity Chromatography

The cultivated cells were harvested by centrifugation at 12,000 rpm for 30 min. After centrifugation, cell pellets were collected and resuspended with 25 ml of buffer A (50 mM Tris, 0.5 mM EDTA at pH 8.0 plus 5% glycerol) per 1 g wet cell pellet. Resuspended cells were disrupted by sonication (Sonics & Materials Inc., CT, USA) and centrifuged at 12,000 rpm for 30 min. After centrifugation, the supernatant solution was collected to obtain the soluble expressed MBP-fusion rRANKL. The collected supernatant was loaded on a 5 ml MBP Trap HP column (GE Healthcare Life Sciences, NJ, USA) equilibrated with 20 ml of buffer A. The column was washed with 10 ml of buffer A and then eluted with 20 ml of buffer B (50 mM Tris, 0.5 mM EDTA, 20 mM maltose monohydrate at pH 8.0 plus 5% glycerol). The eluted MBP-fusion rRANKL was analyzed by SDS-PAGE analysis. Based on the SDS-PAGE analysis, the fractions containing MBP-fusion RANKL were pooled.

Cleavage of MBP-Fusion rRANKL by TEV Enzyme

The eluted MBP-fusion RANKL from MBP affinity column was treated with TEV protease (1 µg TEV protease/ 30 µg protein) in TEV standard buffer (50 mM Tris, 0.5 mM EDTA at pH 8.0 plus 5% glycerol (v/v)) in order to cut the MBP fusion part. The TEV enzyme was purified and supplied in our laboratory. In order to express rTEV protease, we used TEV expression cells containing of pET-TEV vector. After cell culture with LB medium, we purified rTEV protease with His-tag chromatography. We added 380 µg of purified rTEV protease into 1.13 L of sample solution (10 µg/ml) and 5 mM dithiothreitol (DTT). The TEV-treated sample was incubated at room temperature for 48 h. After TEV enzyme reaction, the cleaved rRANKL was analyzed by SDS-PAGE analysis. SDS-PAGE was performed according to the supplier's manual (Bio-Rad, CA, USA). Samples were loaded on a 15% SDS-PAGE gel. The SDS-PAGE gel was stained with Coomassie brilliant blue (Sigma-Aldrich, MO, USA), and stained protein bands were quantified using the ImageJ program (http://imagej.nih.gov/ij).

The Second Chromatography for rRANKL Purification with MBP Affinity Chromatography

We performed dialysis to remove binding maltose from TEV-cleaved MBP. We used the Spectra/Por Dialysis membrane, MWCO 3,500 (Spectrumlabs.com, CA, USA). Dialysis was carried out several times with 5 L of buffer A. The dialyzed TEV-treated sample was again loaded on the 5 ml MBP Trap affinity column. The column was equilibrated with 20 ml of buffer A. After sample loading, the column was washed with 10 ml of buffer A, and then eluted with 20 ml of buffer B. The washed and eluted fractions were analyzed by SDS-PAGE analysis. Based on the SDS-PAGE analysis, the fractions containing RANKL were pooled.

Biological Activity Test of the Purified rRANKL with BMMs

Bone marrow macrophages (BMMs) were plated at a density of 1 × 104 cell/well with macrophage colony-stimulating factor (MCSF, 30 ng/ml) and commercial RANKL (R&D Systems, MN, USA) or the purified RANKL in our laboratory. Cells were incubated for 4 days at 37℃, 5% CO2. After 4 days, cells were fixed with 3.7% formalin for 5 min, permeabilized with 0.1% Triton X-100 for 10 min, and stained with TRAP solution (Sigma-Aldrich, MO, USA) for 10 min. To check the biological activity of rRANKL, multinucleated osteoclasts were observed under a microscope.

 

Results and Discussion

Construction of a rRANKL Vector and Cell Culture for MBP Fusion rRANKL

Modified pMAL-c2x vector containing a TEV cleavage site was used to produce the expression vector, pMAL-RANKL, for soluble MBP-fusion RANKL. To confirm the nucleotide sequences of cloned MBP-fusion RANKL, DNA sequencing analysis was performed and compared with theoretical nucleotide sequences for the TNF domain of murine RANKL. The nucleotide sequences for rRANKL were exactly the same as those of the TNF domain for murine RANKL (data not shown). The expression cells for MBP-fusion rRANKL were cultured at 25℃ after induction. To determine a time course of expression during cell culture, a sample was collected at each time point after induction. The growth curve of the expression cells showed a sigmoid growth pattern. The SDS-PAGE analysis revealed that the expression of MBP-fusion rRANKL was significantly increased after induction. The expression level for MBPfusion rRANKL was increased as time passed, and then it was decreased after 6 h induction. The expression level for MBP-fusion rRANKL was highest with 6 h induction (Fig. 1). The optical density (600 nm) of the cultured cell broth was 2.0 by spectrophotometry. The molecular mass of the expressed MBP-fusion rRANKL was around 61.5 kDa.

Fig. 1.Cell culture and expression of MBP-fusion rRANKL. (A) Cell growth curve of MBP-fusion rRANKL. Cells were cultured for 3 h at 37℃and induced with 1 mM IPTG for 21 h. (B) SDS-PAGE analysis of the expressed MBP-fusion rRANKL. Lane 1: protein marker; Lane 2: the collected cell before induction with IPTG; Lanes 3-7: the collected cells at 1, 3, 6, 12, and 21 h culture after induction.

Purification of rRANKL on MBP Affinity Column

After centrifugation for collecting the cells, 1.86 g of wet weight pellet was obtained from 500 ml of fermentation broth. As seen in Fig. 2A, the expressed MBP-fusion rRANKL was expressed in soluble form. After cell lysis, the expressed MBP-fusion rRANKL was located in the supernatant. The total protein concentration of the MBPfusion rRANKL solution was 200 mg (Table 1).

Fig. 2.SDS-PAGE analysis of rRANKL during purification. (A) The 1st MBP affinity chromatography and TEV protease cutting. Lane 1: protein marker; Lane 2: total cell lysate of induced MBP-fusion RANKL; Lane 3: supernatant solution of cell lysate; Lane 4: pellet of cell lysate; Lane 5: Fraction during loading step on the 1st MBP affinity chromatography; Lane 6: Fraction during washing step on the 1st MBP affinity chromatography; Lane 7: Fraction during elution step on the 1st MBP affinity chromatography; and Lane 8: TEV protease treated sample of MBP-fusion rRANKL from the 1st MBP affinity chromatography. (B) Dialysis and the 2nd MBP affinity chromatography. Lane 1: protein marker; Lane 2: TEV protease treated sample of MBP-fusion rRANKL from the 1st MBP affinity chromatography; Lane 3: After dialysis sample of TEV protease treated sample of MBP-fusion rRANKL from the 1st MBP affinity chromatography; Lane 4: Fraction during loading step on the 2nd MBP affinity chromatography; Lane 5: Fraction during washing step on the 2nd MBP affinity chromatography; and Lane 6: Fraction during elution step on the 2nd MBP affinity chromatography.

Table 1.Purification of rRANKL.

The collected supernatant with MBP-fusion rRANKL was loaded on an MBP affinity column equilibrated with buffer A. The MBP affinity column was washed with buffer A and eluted with buffer B. Based on the SDS-PAGE analysis, purified MBP-fusion rRANKL was eluted with buffer B, as seen in lane 7 (Fig. 2A), whereas most of the impurities flowed through the affinity column during the loading and washing steps. To separate fused MBP from rRANKL, the eluted MBP-fusion rRANKL solution from the MBP affinity column was treated with TEV protease. After the development of appropriate conditions for cleavage, the addition of DTT and reaction at room temperature are necessary for the stability of fusion protein and TEV protease and the cleavage efficiency. The TEV cleavage site between rRANKL and MBP was cut by TEV protease, and we could confirm a perfect cutting site by SDS-PAGE analysis (Fig. 2A lane 8 and 2B lane 2). After cutting with TEV protease, rRANKL protein (19 kDa) and MBP (42.5 kDa) appeared on the SDS-PAGE gel. Moreover, there was a TEV protease band in the SDS-PAGE gel. After the first chromatography with the MBP affinity column and TEV protease cleavage, we could obtain 11.3 mg of protein (Table 1).

The cleaved rRANKL and MBP solution was dialyzed to remove maltose, which was derived from buffer B, and then loaded on the same MBP affinity column to purify the cleaved high-purity rRANKL. If we do not carry out the dialysis step to remove maltose, we do not use the same MBP affinity column. When the cleaved rRANKL and MBP solution was loaded on the MBP affinity column, the cleaved rRANKL was not bound to the functional group of the MBP affinity column, whereas the cleaved MBP was bound to the functional group. The fraction that came through the column during the loading and washing step was the purified rRANKL, and the eluted fraction with buffer B was the cleaved MBP in Fig. 2B. The cleaved rRANKL was separated from cleaved MBP by using the MBP-binding character on the chromatography resin of the MBP affinity column. The purification process is summarized in Table 1. The final amount of purified rRANKL was 2.5 mg from 500 ml of fermentation broth, and its purity was over 95% based on the SDS-PAGE result in Fig. 3.

Fig. 3.SDS-PAGE analysis of the purified rRANKL. Lane 1: Protein marker; Lane 2: The final purified rRANKL after the purification process.

Biological Activity of Purified rRANKL

The biological activity of the purified rRANKL was tested with BMMs and compared with that of commercial RANKL. The specific activity of the purified rRANKL was a little lower than that of commercial RANKL. The activity of purified rRANKL increased as its concentration increased (Fig. 4). Comparing specific activities between purified rRANKL and commercial RANKL, 25 ng/ml of purified RANKL showed almost the same biological activity as 5 ng/ml of commercial RANKL. Even though the purified rRANKL did not have a comparatively high specific activity, its purity was good enough to use as the osteoclast differentiation cytokine.

Fig. 4.Biological activity test of purified rRANKL with BMMs. BMMs were cultured for 4 days with M-CSF (30 ng/ml) and the indicated concentration of the commercial RANKL (R&D Systems) or the final purified rRANKL.

RANKL production in mammalian cells has the disadvantages of high cost and virus contamination [8]. On the other hand, the E. coli expression system has low cost in a high yield, but it is usually expressed as an inclusion body. This problem can be solved by using diverse fusion tagging proteins. MBP has a high solubility and expression level compared with other tagging proteins in a recombinant human erythropoietin study [7]. The modified pMAL-c2x expression vector has the MBP fusion and a TEV protease cleavage site. Thus, we could express rRANKL as a soluble MBP fusion form and purify the expressed rRANKL using an MBP affinity column. To isolate the MBP-fusion rRANKL, the MBP affinity column was used because MBP-fusion protein has high affinity for the MBP affinity column. Thus, we used the first MBP affinity chromatography technique. Additionally, the maltose binding MBP-fusion protein was dialyzed to remove maltose from the cleaved MBP prior to the second MBP affinity column purification after TEV protease cleavage. The second MBP affinity chromatography was carried out to purify the cleaved rRANKL. The two chromatography steps with the same MBP affinity column were very effective and simple to purify the rRANKL. We obtained 2.5 mg of purified RANKL from 500 ml of E. coli fermentation broth. The purity was greater than 95%. The purified rRANKL had biological activity as the osteoclast differentiation cytokine, even though its specific activity was lower than that of commercial RANKL. The overall purification process was more effective and simpler than that in previous reports.