Development of a Protein-Based Sensing System for the Quantitation of Hydroxylated Polychlorinated Biphenyls
Kendrick Turner, Shifen Xu, Patrizia Pasini, Leonidas Bachas, Sylvia Daunert
Polychlorinated Biphenyls (PCBs) have been identified as toxic, persistent pollutants and have been detected in environmental and biological samples including human serum, plasma, and whole blood. Hydroxyl-PCBs (OH-PCBs) are present in the environment as a result of past industrial pollution and are present in living organisms as a product of the metabolism of PCBs and as a result of exposure from the environment. In order to evaluate exposure patterns in both environmental and biological samples, the development of a rapid, portable, selective, and sensitive assay for the detection of these compounds in both environmental and biological samples is advantageous.
To that end, a protein based sensing system for the detection of OH-PCBs is being developed. The protein HbpR from Pseudomonas azelaica acts as a negative regulator for the expression of genes from the hbp operon which allow the microorganism to degrade OH-PCBs and use them as a source of carbon and energy. Upon binding OH-PCBs, HbpR undergoes a conformational change which will be employed in the development of a biosensing system. The protein will either be labeled with a flurophore or prepared as a fusion protein to the Green Fluroescent Protein (GFP). Upon analyte binding and the resulting conformational change, the fluorescence signal from the fluorophore or GFP should change in a dose-dependent fashion, allowing the quantitation of OH-PCBs.
The hbpR gene has been cloned into the pRSETB expression vector as well as the pEGFP expression vector for expression of HbpR and the HbpR-EGFP fusion proteins respectively. The DNA sequence of the resulting expression plasmids has been verified. Preliminary protein expression work has shown expression of HbpR in an insoluble form, and current work is focused on obtaining a properly folded, soluble form of the protein to be used in the development of a biosensing system.
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