Bifunctional fusion proteins for the detoxification and monitoring of organophosphate pesticides: Surface expression and environmentally triggered purification/immobilization

Organophosphorus nerve agents are highly toxic compounds that are widely used as pesticides and chemical warfare agents. Novel bifunctional fusion proteins were created to address the shortcomings of current bioremediation and detection strategies. In one example, whole cell biocatalysts were generated by targeting a detoxification enzyme, organophosphate hydrolase (OPH), onto the cell surface in order to alleviate the potential diffusional limitations created by the cell membrane. Using an ice-nucleation protein (INP) anchor, OPH was successfully localized onto the cell surface of Escherichia coli, Pseudomonas sp. and Moraxella sp., resulting in whole cell biocatalysts that rapidly hydrolyzed organophosphate nerve agents. Utilizing Moraxella sp. and it's natural p-nitrophenol degradative pathway, simultaneous degradation of the pesticide as well as it's hydrolysis product was achieved for complete mineralization of selected pesticides.; A bifunctional fusion protein capable of purifying OPH without the use of conventional chromatography was also described. OPH was fused to a thermally responsive biopolymer, elastin-like protein (ELP). The ELP fusion enabled rapid purification of the OPH fusions with simple environmental triggers. The fusion protein retained nearly identical kinetics as the wild-type OPH. In addition the ELP domain served as an immobilization tag onto hydrophobic supports. By creating a spatially resolved temperature profile, a simple and reversible patterning technique can be achieved on an inexpensive substrate by thermally addressing a patterned hydrophobic surface template.