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Immuno-detection of dioxins using a recombinant protein of aryl hydrocarbon receptor (AhR) fused with sfGFP

BACKGROUND: 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...

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Autores principales: Faiad, Walaa, Hanano, Abdulsamie, Kabakibi, Mohamed Maher, Abbady, Abdul Qader
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4915173/
https://www.ncbi.nlm.nih.gov/pubmed/27328714
http://dx.doi.org/10.1186/s12896-016-0282-9
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author Faiad, Walaa
Hanano, Abdulsamie
Kabakibi, Mohamed Maher
Abbady, Abdul Qader
author_facet Faiad, Walaa
Hanano, Abdulsamie
Kabakibi, Mohamed Maher
Abbady, Abdul Qader
author_sort Faiad, Walaa
collection PubMed
description BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: this highly expressed and functional sfGFP-AhR fusion protein provides a promising molecular tool for detecting and quantifying different congeners of dioxins. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12896-016-0282-9) contains supplementary material, which is available to authorized users.
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spelling pubmed-49151732016-06-22 Immuno-detection of dioxins using a recombinant protein of aryl hydrocarbon receptor (AhR) fused with sfGFP Faiad, Walaa Hanano, Abdulsamie Kabakibi, Mohamed Maher Abbady, Abdul Qader BMC Biotechnol Research Article BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: this highly expressed and functional sfGFP-AhR fusion protein provides a promising molecular tool for detecting and quantifying different congeners of dioxins. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12896-016-0282-9) contains supplementary material, which is available to authorized users. BioMed Central 2016-06-21 /pmc/articles/PMC4915173/ /pubmed/27328714 http://dx.doi.org/10.1186/s12896-016-0282-9 Text en © The Author(s). 2016 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research Article
Faiad, Walaa
Hanano, Abdulsamie
Kabakibi, Mohamed Maher
Abbady, Abdul Qader
Immuno-detection of dioxins using a recombinant protein of aryl hydrocarbon receptor (AhR) fused with sfGFP
title Immuno-detection of dioxins using a recombinant protein of aryl hydrocarbon receptor (AhR) fused with sfGFP
title_full Immuno-detection of dioxins using a recombinant protein of aryl hydrocarbon receptor (AhR) fused with sfGFP
title_fullStr Immuno-detection of dioxins using a recombinant protein of aryl hydrocarbon receptor (AhR) fused with sfGFP
title_full_unstemmed Immuno-detection of dioxins using a recombinant protein of aryl hydrocarbon receptor (AhR) fused with sfGFP
title_short Immuno-detection of dioxins using a recombinant protein of aryl hydrocarbon receptor (AhR) fused with sfGFP
title_sort immuno-detection of dioxins using a recombinant protein of aryl hydrocarbon receptor (ahr) fused with sfgfp
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4915173/
https://www.ncbi.nlm.nih.gov/pubmed/27328714
http://dx.doi.org/10.1186/s12896-016-0282-9
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