Enhanced In Vitro Refolding of Soluble Human Glucocorticoid-Induced TNF Receptor-Related Ligand
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- Kovács, E., Szilágyi, L., Koncz, G. et al. Appl Biochem Biotechnol (2013) 170: 819. doi:10.1007/s12010-013-0232-0
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The glucocorticoid-induced tumor necrosis factor receptor (GITR) is a member of the tumor necrosis factor receptor superfamily. Attachment of GITR to its ligand (GITRL) regulates diverse biological functions, including cell proliferation, differentiation, and survival. In this study, the extracellular region of human GITRL (hGITRL) was cloned, expressed, and purified. The coding sequence of the extracellular region of hGITRL was isolated from human brain cDNA and inserted in pET20b vector. The hGITRL was expressed in Escherichia coli BL21 (DE3) Star at 37 and 25 °C. The majority of the protein was found in inclusion bodies. We identified three important factors for efficient refolding of hGITRL: a ratio of GSH/GSSG, pH, and addition of polyethylene glycol. The renaturated protein was purified by Ni-NTA chromatography. The overall yield of the expression and refolding was higher than 50 mg/l E. coli culture grown at 37 °C. Size exclusion chromatography showed that hGITRL exists as mixture of various multimeric forms in solution. We tested the association of recombinant hGITRL with THP-1 and U937 cell lines and its activity to promote extracellular signal-regulated protein kinase phosphorylation. The results showed that the recombinant protein was biologically active.
KeywordsGITRLHeterologous expressionInclusion bodyPEGSize exclusion chromatography
The tumor necrosis factor (TNF) superfamily members are regulators of cell proliferation, differentiation, and survival . The glucocorticoid-induced tumor necrosis factor receptor (GITR) is member of the TNF receptor superfamily (TNFRSF); it was originally cloned in a glucocorticoid-treated hybridoma T cell line . GITR is a type I transmembrane protein with a cysteine-rich extracellular domain and is also known as AITR (activation-inducible TNFR family member) or TNFRSF18 [3, 4]. In mice, expression of GITR has been detected in regulatory T cells, B cells, natural killer (NK) cells, granulocytes, and macrophages [5–7], whereas in humans, GITR expression has been described in macrophages and NK cells [8–10]. Effector CD4+ and CD8+ T cells express GITR at low levels but rapidly upregulate GITR expression upon activation . GITRL is the natural ligand of GITR [3, 6] and is expressed on the surface of various antigen-presenting cells, including macrophages, B cells, and immature and mature dendritic cells [5, 7]. Addition of soluble GITRL or anti-GITR antibody arrested the suppressive activities of CD4+CD25+ regulatory T cells [11, 12].
Human GITRL (hGITRL) expression was detected in CD8 cells, and expression level was increased in CD8 cells from tumor-positive lymph node of breast cancer patient . Tumor cell lines and patient leukemia cells express substantial levels of GITRL, and elevated levels of soluble GITRL (sGITRL) are present in sera of patients with various cancers [8, 10, 14]. NK cells contribute to cancer immunosurveillance; GITR triggering diminishes NK cell effector function . Addition of sGITRL-containing patient sera to cocultures of NK and tumor cells reduced NK cell cytotoxicity. Neutralization of sGITRL using a GITR-Ig fusion protein restored NK cell reactivity . It was also described that GITR/GITRL system plays a key role in both acute and chronic inflammation [15–17]. Recent studies show that GITR and GITRL expression increases in macrophages from human atherosclerotic plaques , rheumatoid arthritis , in lymphocytes from rheumatoid arthritis patients , and stimulation of GITRL-induced expression of proinflammatory cytokines in synovial macrophages . GITRL expression also increases in patients with systemic lupus erythematosus . In light of these findings, it is likely that an efficient method for the preparation of recombinant hGITRL would open alternative pathways for some human disease detection and for monitoring of efficacy for the use of GITRL in various therapies.
In the present paper, we described an efficient procedure for refolding of the hGITRL form inclusion body.
Materials and Methods
Construction of the pET20b-hGITRL Recombinant Vector
Human brain cDNA was a generous gift from Dr. Júlia Tóth . The putative extracellular portion of hGITRL (amino acid positions 56–177) from human brain cDNA was amplified by polymerase chain reaction. The forward primer 5′- GGCATATGGAGCCCTGTATGGCTAAGTTTGGACC −3′ contained an NdeI restriction site at the 5′ end of the gene, and the reverse oligonucleotide 5′-GGAGGATCCTGGGAGATGAATTGGGGATTTGC-3′ contained a BamHI restriction site at the 3′ end of the coding sequence of the hGITRL. The amplified fragment was inserted in pET20b expression plasmid producing recombinant vector pET20b-hGITRL. The plasmid encodes a His6-tag, which promotes the purification of the recombinant protein by affinity chromatography.
Production of the hGITRL in Escherichia coli BL21 Star (DE3)
The recombinant plasmid was transformed into chemically competent E. coli BL21 (DE3) Star cell. The transformed cell suspension was plated on Lysogeny broth (LB) agar containing 100 μg/ml ampicillin supplemented with 1 % glucose and incubated overnight at 37 °C in order to select transformed colonies.
Starter culture of single colony was obtained by overnight incubation at 37 °C, at 250 rpm shaking in LB ampicillin supplemented with 1 % glucose. Expression culture was set up in volumes of 250 ml in LB media supplemented with 100 μg/ml ampicillin. The culture was grown at 37 °C to an OD600 value of about 0.8–0.9, isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM, and the culture was incubated for 4 h at 37 °C. The cells were harvested by centrifugation (5,000 rpm, 10 min, 4 °C) and lysed by sonication. The results of the expression were analyzed by 16.5 % sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels and visualized by Coomassie Blue staining.
Solubilization and Refolding of the Inclusion Bodies
The inclusion body obtained from 250 ml culture grown at 37 °C was solubilized overnight at room temperature in 5 ml 6 M guanidine hydrochloride (50 mM Tris–HCl buffer, pH 8.8, and 1 mM dithiothreitol). The solubilized inclusion body was centrifuged at 14,000 rpm for 10 min, filtered (0.22 μm, Millipore), and stored at 4 °C. Protein concentration was determined by using the Quick Start Bradford Protein Assay kit (Bio-Rad) using bovine gamma-globulin standard.
Factorial screening for optimal conditions of refolding was performed in a final volume of 1 ml, following a modified protocol described previously . The final concentrations of components in the refolding buffers are as follows: 0.1 M MES, pH 6.0; 0.1 M Na-phosphate, pH 7.0; 0.1 M Tris–HCl, pH 8.0; 0.1 M Tricine, pH 9.0; and 0.05 % polyethylene glycol (PEG-3350). Concentration of reduced and oxidized glutathione was either 5 and 0.5 mM or 0.5 and 5 mM. To 1 ml refolding buffer, 50 μl of solubilized protein was added at a final concentration of 0.16 mg/ml. After dilution, the samples were mixed on a shaker overnight at 4 °C and then centrifuged at 14,000 rpm for 10 min. Protein concentration in the supernatant was evaluated from optical density at 280 nm using the calculated molar extinction coefficient, ε = 2.54 × 104 M−1 cm−1. Absorbance values were corrected with the readings of appropriate buffers. On the basis of the screen results, the protein refolding was repeated in a final volume of 60 ml (2 ml of the solubilized material was diluted into 60 ml ice-cold refolding buffer). The composition of the refolding puffer, 0.8 M Gu–HCl; 0.1 M Tris–HCl, pH 8.0; 5 mM reduced glutathione; and 0.5 mM oxidized glutathione, was supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma) and 0.05 % PEG-3350.
The refolded protein was purified by adsorption on a Ni-charged polymer matrix (Profinity IMAC Ni-charged Resin, Bio-Rad). The nonspecifically bound proteins were washed with 50 mM sodium phosphate buffer, pH 8.0, containing 500 mM NaCl. Elution of the protein was carried out by 50 mM sodium phosphate buffer, pH 8.0, containing 300 mM NaCl and 250 mM imidazole. Imidazole was removed by dialysis against 20 mM sodium phosphate buffer, pH 8.0, and supplemented with 100 mM NaCl. The protein was stored at 4 °C.
To about 50–100 μl of dialyzed GITRL preparation, 1 ml of 10 % acetonitrile, 0.1 % trifluoroacetic acid was added, and the sample was loaded onto a Vydac C8 reverse-phase column (208TP54). The chromatography was performed by using an Agilent HP 1100 system at room temperature. Flow rate was 0.75 ml/min; buffer A contained 0.1 % (v/v) trifluoroacetic acid in water, and buffer B was 0.09 % trifluoroacetic acid in acetonitrile. After an initial wash with 10 % B, a linear gradient from 10 to 90 % B was applied in 8 min.
Gel filtration analysis was performed using a Superdex-75 column on a fast protein liquid chromatography system (ÄKTA, GE Healthcare Life Sciences) at a flow rate of 0.5 ml/min with buffer composed of 10 mM Na-phosphate, pH 8.0, and 100 mM NaCl. The protein elution was monitored at 280 nm, and the fractions were analyzed by electrophoresis on a 16.5 % SDS-PAGE gel. The column was calibrated with molecular weight marker proteins thyroglobulin (667 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), and myoglobin (17 kDa), and vitamin B12 (1.35 kDa), and a graph was drawn by plotting the partition coefficient Kav on the x-axis and log molecular weight on the y-axis. From this calibration graph, the molecular weights of the eluted proteins were calculated.
The hGITRL was biotinylated using the EZ-Link Sulfo-NHS-LC-Biotinylation kit according to the protocol provided by the manufacturer (Pierce). The biotinylation reaction was stopped by adding 10 mM diethanolamine and then was verified by western blot analysis. The polyvinylidene difluoride (PVDF) membrane (Immobilon-P; Millipore) was blocked with 1× Tris-buffered saline (TBS)–Tween for 1 h and then treated with ExtrAvidin peroxidase conjugate (Sigma, dilution of 1:3,000) for 1 h at room temperature with shaking. The membrane was washed using a solution containing 20 mg diaminobenzidine, 25 μl H2O2, and 50 μl 100 mM NiCl2 in 50 ml TBS.
Flow Cytometric Analysis
Flow cytometric analysis was performed on the FACSCalibur system (Becton-Dickinson, Mountain View, CA). Cells (2 × 105) were washed with PBS containing 0.5 % BSA and 0.1 % sodium azide and incubated with 5 μg of recombinant hGITRL for 1 h on ice. Cells were washed twice and incubated with 0.3 μg of avidin-FITC (Alexa Fluor, Invitrogen) for 20 min. For detection of anti-GITR-PE (Miltenyi Biotec) binding, following preincubation with 5 μg of human IgG for 5 min, the cells were labeled with 2 μg of anti-GITR-PE for 20 min.
Extracellular Signal-Regulated Protein Kinase Phosphorylation Assay
A total of 2 × 106 THP-1 cells were treated with hGITRL (10 μg/ml) for 10 and 30 min at 37 °C. The cells were harvested by centrifugation and boiled in 100 μl of reducing Laemmle buffer, and 25–25 μl sample was loaded onto two 10 % SDS-PAGE. After separation, proteins were transferred to a PVDF membrane. The membranes were incubated either with the anti-phospho-extracellular signal-regulated protein kinase (ERK)1/2 (Thermo Scientific, dilution of 1:1,000) or anti-ERK1/2 (Cell Signaling Technology, 1:1,000) for overnight at 4 °C. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, 1:5,000) for 1 h. The proteins of interest were detected by using the Chemiluminescent Substrate kit (Thermo Scientific).
Results and Discussion
E. coli is frequently used as the host for protein production; however, the target proteins often accumulated in the form of inclusion bodies . Some recombinant proteins appear in both the soluble and insoluble cell fractions, and many others are only produced as inclusion bodies (IBs) [25–27].
The proteins in IBs are not biologically active, so refolding of proteins is necessary. After isolation, inclusion body was solubilized in 6 M guanidine hydrochloride supplemented with 1 mM DTT. The solubilized inclusion body was centrifuged, filtered, and used for refolding screen as described in “Materials and Methods.” The aim of this screen was to obtain the optimal pH and ratio of the reduced and oxidized glutathione during refolding. We also tested the effect of PEG-3350 during refolding of hGITRL . Only one concentration of each additive was studied in the refolding screen.
Refolding was initiated by rapid dilution of the solubilized protein into 20-fold excess of appropriate buffer solution. After overnight shaking, the precipitate was removed by centrifugation. The efficacy of refolding was estimated on the basis of the buffer-corrected absorbance of the supernatants at 280 nm using the calculated molar extinction coefficient (ε = 2.54 × 104 M−1 cm−1). Under our experimental conditions, A280 = 0.25 corresponded to 100 % refolding.
In summary, we cloned and expressed the extracellular region of the hGITRL and determined optimal conditions for its refolding. We identified three important factors for the refolding of hGITRL: a ratio of GSH/GSSG, pH, and addition of PEG. We have shown that refolded recombinant hGITRL exists in solution as mixture of various multimeric forms, probably dimers and trimers. Using flow cytometric analysis, we demonstrated that the refolded hGITRL could bind to GITR of U937 and THP-1 cell lines. In vitro stimulation of this cell lines with recombinant hGITR ligand induced phosphorylation of ERK. From these results, we concluded that hGITRL was in its proper conformation. The obtained recombinant hGITRL would provide a way for further in vitro studies on the relation between GITR and GITRL in the antitumor immune response and inflammation.
This work was supported by the Department of Bioengineering, Sapientia Hungarian University of Transylvania; the Department of Biochemistry of Eötvös Loránd University; and the Department of Immunology, University of Debrecen.