Immuno-detection of dioxins using a recombinant protein of aryl hydrocarbon receptor (AhR) fused with sfGFP.
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.
Department of Animal Biology, Faculty of Sciences, Damascus University, Damascus, Syria.
Walaa
W
Faiad
Division of Toxicology, Department of Molecular Biology and Biotechnology, Atomic Energy Commission of Syria (AECS), Damascus, Syria.
Abdulsamie
A
Hanano
Department of Animal Biology, Faculty of Sciences, Damascus University, Damascus, Syria.
Mohamed Maher
MM
Kabakibi
Division of Microbiology and Immunology, Department of Molecular Biology and Biotechnology, Atomic Energy Commission of Syria (AECS), Damascus, Syria. aabady@aec.org.sy.
Abdul Qader
0000-0002-3898-5269
AQ
Abbady
D001681:Biological Assay / Q000379:methods; D015374:Biosensing Techniques / Q000379:methods*; D004147:Dioxins / Q000032:analysis* / Q000276:immunology; D004926:Escherichia coli / Q000276:immunology; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000276:immunology*; D007118:Immunoassay / Q000379:methods*; D008163:Luminescent Measurements / Q000379:methods*; D018336:Receptors, Aryl Hydrocarbon / Q000737:chemistry / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D004147:Dioxins; D018336:Receptors, Aryl Hydrocarbon; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins
AhR; Cloning; Fusion proteins; Protein expression; TCDD; sfGFP