Immuno-detection of dioxins using a recombinant protein of aryl hydrocarbon receptor (AhR) fused with sfGFP
© The Author(s). 2016
Received: 7 April 2016
Accepted: 13 June 2016
Published: 21 June 2016
Dioxins are one of the most toxic groups of persistent organic pollutants. Their bioaccumulation through the food chain constitutes a potential risk for human health. Upon cell entry, dioxins bind specifically and firmly to the aryl hydrocarbon receptor (AhR), leading to the stimulation of several enzymes responsible for its detoxification. Dioxin/AhR interaction could be exploited as an affordable alternative to a variety of analytical methods for detecting dioxin contamination in the environment.
In this work, the ligand binding domain (LBD) of the AhR was cloned downstream a superfolder form of the green fluorescent protein (sfGFP), resulting in the construct pRSET-sfGFP-AhR. High level of expressed sfGFP-AhR fusion protein (50 kDa) was recovered from the inclusion bodies of E. coli by simple solubilization with the Arginine, and purified by affinity chromatography via its N-terminal 6 × His tag. Its purity was confirmed by SDS-PAGE analysis and immunoblotting with anti-His or anti-GFP antibodies. Indirect ELISA revealed the ability of the sfGFP-AhR, but not the sfGFP, to bind to the immobilized dioxin with the possibility to detect such interaction by both its 6 × His and GFP tags,Competitive ELISA showed that anti-dioxin antibody was more sensitive to low dioxin concentrations than sfGFP-AhR. Nevertheless,the detection range of sfGFP-AhR fusion was much wider and the detection limit was of about 10 ppt (parts per trillion) of free dioxin in the tested artificial samples.
this highly expressed and functional sfGFP-AhR fusion protein provides a promising molecular tool for detecting and quantifying different congeners of dioxins.
Collectively termed as dioxins, polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) are the most toxic group of the persistent organic pollutants (POPs). Depending on the chlorination level (P = 1–8), dioxins form a generic group of 75 PCDD congeners and 135 PCDF congeners with varying degrees of toxicity. The congeners with chlorine atoms substituted in the lateral 2, 3, 7 and 8 positions of the aromatic rings are considered as the most toxic. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), with a toxic equivalency factor (TEF) of 1.0, is the most toxic congener of dioxins . Consequently, TCDD was used as a good candidate for investigations of the physiological and toxicological effects of this class of chemicals [2, 3].
Dioxins are a major product of industrial, municipal and domestic incineration and combustion processes  and during industrial processes involving chlorinated aromatic and aliphatic compounds, such as pesticides and herbicides synthesis . Currently, incineration of the solid wastes contributes the most to the release of these compounds into the environment .
Due to their persistence and high lipophilicity, PCDD/Fs are subject to lipophilic bio-concentration and accumulation in the food chain . Their adverse health effects are now well reported; for example the wasting syndrome , immunotoxicity , teratogenicity , dysfunctional immune and reproductive systems  and carcinogenesis . An effective and serious contamination with dioxins has been previously reported in soils, sediments, air and water [11, 12]. In this context, numerous analytical techniques have been developed for the detection and quantification of dioxins. High resolution gas chromatography combined with high resolution mass spectrometry (HRGC/HRMS) is considered the most reliable and sensitive method for separation, identification and determination of the congener-specific concentrations of dioxins . However, this technique requires a sophisticated platform and highly trained personnel, besides being very costly and time-consuming. Consequently several rapid and inexpensive screening assays have been proposed. Immuno-detection, which depends on the ability of a certain compound to be precisely recognized and bound by a specific antibody, makes the base of what is known as the enzyme-linked immunosorbent assay (ELISA) [14, 15]. Alternatively, the detection of signal transduction effects that are related to dioxin stimulation in vitro or in cells has been exploited using sophisticated methods; such as the chemical activated luciferase gene expression (CALUX) system .
The biological effects of the dioxins in the body critically depend on a cytosolic protein, the aryl hydrocarbon receptor (AhR) . The AhR resembles nuclear receptors (among which it was initially categorized) acting as a ligand-activated transcription factor . In response to activation by dioxins, AhR signalling pathway modifies the expression levels of numerous genes. The best characterized of these pathways at the molecular level is that leading to the induction of the gene for a Phase I drug-metabolizing enzyme; CYP1A1 . In the dormant state, the AhR resides in the cytosol in a protein complex also containing a dimer of heat shock protein 90 (HSP90), an AhR-associated protein-9 and p23 . These chaperone proteins stabilize the AhR maintaining it in a conformation that is unable to enter the nucleus but optimal for ligand binding . The AhR then dissociates from the chaperones and heterodimerizes with the AhR nuclear translocator (ARNT) . AhR/ARNT dimer binds to the major groove of the DNA helix at specific sites . Structurally, the AhR belongs to basic Helix–Loop–Helix/Periodic, AhR nuclear translocator, single-minded (bHLH/PAS) proteins. The AhR contains several important structural elements; including an N-terminal bHLH domain, a C-terminal transcriptional activation domain and a central PAS domain containing two degenerated repeats (PAS-A and PAS-B). The ligand binding domain (LBD) of the AhR is localized between amino acids 230 and 397 , and has a similar affinity for TCDD binding as the full-length AhR .
In line of these insights we investigated the possibility to fuse the LBD of the AhR with a marker protein, the green fluorescent protein (GFP), in order to exploit the resulting recombinant fusion protein in dioxins detection. The GFP from Aequorea jellyfish is widely used as an excellent expression tag for several fusion proteins , and it has been expressed in a variety of species; including bacteria, plants, Drosophila melanogaster, Caenorhabditis elegans, zebrafish, andmammals [24–26]. Waldo and co-workers reported the engineering of an enhanced superfolder (sf) form of the GFP, and this sfGFP showed increased resistance to denaturation, improved folding kinetics, and increased resistance to aggregation during protein expression . Therefore, the main objective of the current study was to express the sfGFP-AhR fusion protein in E. coli. Computational 3D structure prediction of this fusion suggested a total accessibility of the AhR moiety for TCDD binding, and consequently this was confirmed in our immunological experiments. Optimized sfGFP-AhR fusion could effectively be invested in as an affordable, rapid, sensitive and environment-friendly technique to detect the presence of dioxins in environment samples and food commodities.
Preparation of the AhR cDNA from HepG2 cell line
Total RNA was extracted from the human hepatoma cells (HepG2) using IllustraRNAspin Mini Kit (GE Life Sciences) according to the manufacturer's manual. 2 μg of the RNA was reverse-transcribed to the cDNA using Ready-to-Go You-prime first-strand-beads (GE Life Sciences) with oligo-dT15–18 (Invitrogen). 2 μl of the cDNA was used as a template in a PCR with a pair of specific primers for the AhR(LBD); AhR(BamHI)-F and AhR(EcoRI)-R (Additional file 1: Table S1). Primers were designed to amplify the LBD of the AhR gene and to add BamHI and EcoRI restriction sites at the 5' and 3' ends, respectively. The fragment of LBD was amplified by a high fidelity Taq DNA polymerase (AccuPrime™ Kit; Invitrogen) at 55 °C annealing temperature. Amplified AhR fragment and the pRSET-sfGFP plasmid  were digested with BamHI and EcoRI (Fermentas) then ligated using the T4 DNA ligase following standard procedures (Fermentas).
Bacterial strains and growth conditions
E. coli strains TOP10 (Invitrogen) and BL21(DE3) Gold (Novagen) were used in cloning and protein expression. E. coli was grown in Luria Broth (LB; 1 % Tryptone, 0.5 % yeast extract, 171 mM NaCl) (Bio Basic Inc.) with ampicillin antibiotic (100 μg/ml, Sigma) in orbit-rotating incubator at 37 °C. Freshly prepared electro-competent E. coli TOP10 cells were transformed with the plasmid constructs by electroporation. Colony PCR screening for positive AhR clones was performed using pRSET specific primers (T7F/T7R) (Additional file 1: Table S1). The plasmid constructs were isolated from some positive clones by plasmid Miniprep Kit (Qiagen) after being grown in LB/ampicillin medium. The identity of these plasmid constructs was confirmed by digestion with the restriction enzymes and by sequencing using different sequencing primers covering the full-length LBD (Additional file 1: Table S1).
Expression of the sfGFP-AhR fusion protein as inclusion bodies
The confirmed plasmid constructs; the pRSET-sfGFP and pRSET-sfGFP-AhR, were used to transform E. coli BL21 (DE3) Gold cells by electroporation. Protein expression was performed in 250 ml shake flasks by growing the bacteria in LB medium till an optical density of 0.5 to 0.7 was reached, and then the expression was induced with 0.5 mMIsopropylthio-D- galactoside(IPTG, Promega) for 16 h at 15 °C. After centrifugation, the pellet of bacteria was resuspended on the binding buffer(50 mMtris-HCl pH 8.0, 150 mM NaCl and 1 mMphenylmethylsulfonyl fluoride (PMSF, Sigma) supplemented with a complete protease inhibitor cocktail (Roche), then lysed by 8 pulses (15 s each) of sonication at 22 % amplitude (750-Watt Ultrasonic Homogenizer, Cole-Parmer) in ice. The lysate containing soluble proteins as the sfGFP was cleared by centrifugation at 12000 × g for 20 min at 4 °C. The pellet of the inclusion bodies containing insoluble proteins (as the sfGFP-AhR) was re-suspended by sonication in the same binding buffer supplemented with (5 mM DTT, 5 mM EDTA, 1 M urea and 1 % triton-x). This washing step is designed to remove contaminants, especially proteins that may have adsorbed onto the hydrophobic inclusion bodies during processing and could affect protein refolding yield. After washing with the basic binding buffer to eliminate the detergents, inclusion bodies were ready to be solubilized using the different reagents.
Refolding and solubilizion of sfGFP-AhR inclusion bodies
To assess the optimal reagent for assisting the correct refolding of the sfGFP-AhR, inclusion bodies were prepared at 0.1 mg/ml TBS buffer (20 mMtris-HCl pH 8.0 and 150 mM NaCl). 500 μl of this preparation was distributed into several 1.5 ml microtubes then centrifuged at max speed for 5 min. The pellets in the microtubes were dispersed and dissolved by sonication in the same buffer containing different concentration of various additives reported in the literature  (Additional file 2: Table S2). After 30 min of incubation at RT, supernatant (soluble fraction) was recovered from each condition by centrifugation and the remaining pellet of insoluble sfGFP-AhR was dissolved by sonication in 500 μl of 8 M urea-containing buffer (insoluble fraction). The formation of a fluorophore in the soluble and insoluble fractions was monitored by measuring the fluorescence density, expressed as a relative fluorescent unit (RFU), at excitation wavelength 485 nm with emission wavelength 538 nm (Fluoroskan Ascent, Thermo Labsystems), and the fluorophore fluorescein (Sigma) was used as a control.
Purification of the sfGFP and sfGFP-AhR fusion proteins
Soluble fractions of E. coli extract containing the sfGFP and Arginine-refolded sfGFP-AhR were dialysed in binding buffer containing 20 mM imidazole and then purified using a 5 ml column of nickel charged nitrilotriacetic acid (NTA) superflow sepharose (Qiagen) installed on the fast protein liquid chromatography (FPLC) AKTA prime plus system (GE Life Sciences). After washing, bound proteins were eluted from the column with 500 mM imidazole-containing buffer. The eluted fraction was concentrated on Vivaspin concentrators with a molecular mass cut-off of 10 kDa (Vivascience). The concentration of the purified proteins was determined by the Bradford method, and their purity was evaluated in SDS-PAGE prepared using stacking gel 5 % and running gel 12 %. After electrophoresis, the gel was stained in coomassie brilliant blue buffer (45 % methanol, 10 % acetic acid and 0.25 % coomassie R250) for 2 h and then washed several times in the destaining buffer.
Immunoblotting of the sfGFP and sfGFP-AhR
Purified proteins were separated by SDS-PAGE before blotting onto 0.45 μm nitrocellulose membranes (Bio-Rad) using 1× transfer buffer (25 mMtris-HCl, 200 mM glycine, 0.1 % SDS and 20 % methanol). After blotting, the membranes were washed with TBS-T (0.05 % Tween-20 in TBS) then incubated in the blocking buffer (5 % skimmed milk in TBS-T). The membranes were incubated with 1:2000 dilution of either mouse anti-GFP (Roche) or mouse anti-6 × His (R&D Systems) antibodies for 1 h at RT. After several washes with TBS-T, the membranes were incubated with (1:2000) dilution of a secondary goat anti-mouse antibody conjugated to the horseradish peroxidase (HRP, Bethyl laboratories) for 1 h at RT. Bands revelation was achieved by adding 3-amino-9-ethylcarbazole (AEC, Sigma) chromogen substrate in acetate buffer in the presence of a hydrogen peroxide.
Indirect ELISA assay for testing sfGFP-AhR
An indirect ELISA was performed by immobilizing the antigens (10 nM), sfGFP-AhR or sfGFP, on a standard MaxiSorp 96-well plate (Nunc) overnight in a bicarbonate/carbonate coating buffer (100 mM, pH 9.6). Alternatively, dioxin binding was tested using a TCDD-pre-coated 96-well ELISA microplate (Abraxis LLC). Before use, all different plates were incubated in the blocking buffer (5 % skimmed milk in TBS-T) for 2 h at RT then washed 3 times with TBS-T. Immobilized TCDD in the wells was directly detected using a rabbit-anti-TCDD (Abraxis), following the manufacturer's instructions. Otherwise, the sfGFP-AhR and sfGFP were diluted to the desired concentration (100 nM) in 1 % blocking buffer then applied to the wells (100 μl) for 16 h at 4 °C, then washed 3 times with TBS-T. Antigens (immobilized or bound to TCDD) were detected by an incubation for 1 h at RT with a diluted (1:2000) mouse anti-6 × His, rabbit anti-6 × His (Bethyl laboratories), mouse anti-GFP or rabbit anti-GFP (a homemade product, ). After 5 washes, diluted (1:2000) goat anti-mouse or goat-anti-rabbit IgGs conjugated to horseradish peroxidase (HRP, Bethyl laboratories) were added to the appropriate wells. The wells were then washed 5 times before bound conjugates were detected with 50 μl of 3,3′,5,5′-tetramethylbenzidine (TMB, Sigma) ready-to-use substrate. The reaction was finally stopped with the addition of 50 μl of 1 M H2SO4. The spectroscopic absorbance of each well was measured in an automated plate reader at a wavelength of 450 nm.
TCDD detection by a competitive indirect ELISA using the sfGFP-AhR
Using the same TCDD-pre-coated plates and doing the same primary steps of blocking and washing, the competitive ELISA was carried out by incubating the sfGFP-AhR (30 nM) or rabbit-anti-TCDD with serial dilutions (from 0 – to 100,000 parts per trillion (ppt) in 1 % DMSO-containing blocking buffer) of free TCDD (stock at 10 μg/ml, purity 99 %, Supelco Inc.) in 100 μl volume for 2 h at RT. Different conditions were then transferred to TCDD-pre-coated wells and incubated for further 2 h at RT. The following steps were performed as described earlier using a rabbit-anti-GFP antibody, goat-anti-rabbit-HRP and the TMB substrate. The experiments were performed three times to determine the mean values (used to draw the graphs) and the error bars were calculated by dividing the standard deviation of the data by the square root of number (3) of values that make up the mean.
Design of the sfGFP-AhR fusion protein
Cloning of AhR gene into the pRSET-sfGFP plasmid
Plasmids like the pRSET with a T7 promoter are widely used for high throughput protein expression in E. coli strains carrying the lambda DE3 lysogen  (Additional file 4: Figure S2 and Additional file 2: Table S2).
Expression of the fluorescent sfGFP-AhR fusion protein
Optimization of in vitrosfGFP-AhR refolding
Purification and immunodetection of the sfGFP-AhR
Detection of the interaction of the sfGFP-AhR with dioxin
To date, many reports have described the heterologous expression of the recombinant AhR in both prokaryotic and eukaryotic cell types. Particularly, the recombinant expression of the ligand-binding competent AhR would have significant advantages for many bioanalytical applications regarding dioxins detection. The majority of the described methods take advantage of the ability of dioxin/AhR complexes to trigger certain signal transduction pathways . Transgenic tobacco plants carrying the recombinant mouse AhR-mediated β-glucuronidase (GUS) reporter gene expression system was designed and showed a significantly increased GUS activity when treated with certain AhR ligands . Such assays did not need any extraction and purification of chemicals and seemed to be useful for a biochemical assay of dioxins and dioxin-like compounds . Furthermore, recombinant yeast cells Saccharomyces cerevisiae have been designed as a dioxin bioassay system, in which the expressed full length human or mouse AhR/Arnt complexes form after ligand binding and interact with a responsive element located on the promoter of ß-galactosidase reporter gene , and this system was utilized to identify potent endogenous AhR ligands from human urine and bovine serum . Baculovirus expression vector system was utilized for high level production of the full-length human and rat AhR in Spodopterafrugiperda (Sf9), and the recombinant proteins were active in the quantitative analysis of the ligand . In fact, the absence of the disulfide bridges and glycosylation sites in the LBD of AhR makes it a more suitable target for expression in E. coli. However, several attempts for LBD expression in E. coli were unsuccessful because of its instability and its high tendency to aggregate because of misfolding or incomplete folding processes . Therefore, fusion protein technology is considered one of the best solutions to enhance the protein expression and solubility. For example, it chaperones the proper folding, facilitates the purification and reduces the protein degradation or toxic effects . Many fusion moieties are available to improve the expression and purification of proteins, including glutathione S-transferase (GST), maltose-binding protein (MBP), thioredoxin A (TrxA), and the GFP .
In the current study, we successfully produced the LBD of AhR in a fusion with the sfGFP, resulting in its expression at a high level (~150 mg/L of bacteria culture) in the cytoplasm of E. coli. Despite its well documented stability and solubility, the enhanced form of the GFP, the superfolder GFP, failed to maintain the correct folding of the expressed AhR, leading therefore to its accumulation into the inclusion bodies. This was disappointing since many reports described the recovery of several insoluble proteins from the inclusion bodies simply by their merging with the sfGFP, e.g. the human growth hormone , and in certain cases, this resulted in the enhancement of their physiological roles . The culture and induction conditions, including temperatures and IPTG concentrations, may play a critical role in the solubility of many recombinant proteins, especially, when some stringent culture conditions provoke the production of large quantities of the exogenous protein that the cell folding machinery fails to process . Hence, several conditions were tested to assess their influence on the solubility of the sfGFP-AhR which was simply monitored by comparing the colour of the bacterial lysate and precipitate. Unfortunately, the fusion greenish protein was always found in the inclusion bodies regardless of the conditions used, even with the mild ones; Tm 10 °C and 0.1 mM IPTG, on the contrary, free sfGFP was always soluble when expressed from the plasmid pRSET-sfGFP, using many different culture conditions (data not shown).
Arginine has long been used as an interesting additive for solubilizing the inclusion bodies and enhancing protein solubility and stability . Its mechanism of action relies on inhibiting the aggregation of misfolded proteins and surrounding their exposed hydrophobic surfaces. Furthermore, 0.5–2MArginine, can be used to actively extract the folded proteins from insoluble pellets obtained after lysing E. coli cells . Comparing several tested solubilizing reagents reported in the literature , we found that low concentrations of Arginine, e.g. 0.1 M, were enough to refold totally sfGFP-AhR from the inclusion bodies, and even without a prior step for solubilizing the inclusion bodies in 6 M guanidine-HCl or 8 M urea. Lauroylsarcosine showed a similar effect on the inclusion bodies of the sfGFP-AhR, however its severity, as a strong ionic detergent, compared to the Arginine which is barely a stabilizing amino acid, makes its uses unfavourable in the production procedure of the sfGFP-AhR. Nevertheless, we speculated from this experiment that the structure of the sfGFP-AhR is moderately misfolded, making its recovery from the inclusion bodies an easy and inexpensive task to perform. This could be related to the sfGFP owing to the folding reputation it has, beside, its visible and florescent signals which made it possible to track the transformation of the fusion protein between the insoluble and soluble states, especially when different solubilizing additives were used. Alternatively, the aggregation problem of the AhR can be probably overcome by using eukaryotic heterologous expression systems such as animal or plant cells.
The ability ofsfGFP-AhR to bind to TCDD was proven by a standard and a competitive ELISA. This last type of ELISA is mainly used for the detection of very-small antigens that are difficult to be immobilized on a solid support, hard to purify and composed of a few number of epitopes, e.g. the haptens . It is usually performed to quantify TCDD contamination in water, soil and sediment samples . Despite the fact that anti-TCDD antibody showed more sensitivity for TCDD in competitive ELISA than the sfGFP-AhR, the detection range of the sfGFP-AhR was much wider. According to the USA-Environmental Protection Administration (EPA), the maximum limits for dioxins in wastewater are set to be 5 pg I-TEQ per liter for new companies and 10 pg I-TEQ per liter for existing companies. These ranges which equal 5 to 10 ppt of total dioxins are being covered by estimated sensitivity range of the proposed sfGFP-AhR detection system. For enhancing the affinity, replacing the LBD in the sfGFP-AhR fusion, originated from human AhR, with the equivalent domain from mouse or rat AhR can be suggested, because they are known to have better binding kinetics towards TCDD . Nevertheless, this sfGFP-AhR-based immunoassay can be exploited as a method for analyzing large number of samples, from different sources, for contamination with TCDD, or any similar compounds. An estimated value of TCDD content can be attributed to the measured samples simply by comparing the logarithmic curve fit of the known concentrations of standard TCDD. Samples that are determined to be positive in this immunoassay can undergo congener-specific analyses by HR-GC/MS. Beside its analytical uses, the fluorescent sfGFP-AhR fusion can be used as a powerful method for bio-tracing of TCDD within the cells or tissues in diverse unexploited areas of research. For example, this bioanalytical method will surely contribute to a better understanding of the plant/dioxin relationship and therefore in deciphering the mechanisms involved in plant responses to dioxin exposure [45, 46].
In conclusion, dioxin binding domain of the AhR was produced in a fusion with an enhanced form of the GFP using an efficient E. coli protein expression system. After Arginine-refolding and affinity purification, the sfGFP-AhR fusion protein was able to bind to TCDD using a standard and competitive ELISA, and the detection limit of free TCDD in samples could reach very low levels (~10 ppt).
AhR, aryl hydrocarbon receptor; bHLH/PAS, basic helix-loop-helix/Per-ARNT-Sim; GFP, superfolder green fluorescent protein; LBD, ligand binding domain; PCDDs, polychlorinated dibenzo-p-dioxins; PCDFs, polychlorinated dibenzofurans; TCDD, 2,3,7,8-Tetrachlorodibenzo-p-dioxin sf.
The authors would like to thank the Director General of the Atomic Energy Commission of Syria and the head of the Molecular Biology and Biotechnology department for their continuous support throughout this work.
This work was performed without any external funding.
Availability of supporting Data
The data set(s) supporting the results of this article is(are) included within the article (and its additional file(s)).
WF, carried out all experimental work. AH, designed TCDD-affinity experiments, participated in writing the manuscript. MMK, read and commented the manuscript. AA, led the work, designed all experiments, and wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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