First Name:
Jared
Last Name:
Harris
Major Department:
Chemistry
Thesis Director:
Libby Puckett
Date of Thesis:
May 2012
This thesis project’s purpose was to examine the feasibility of using a fusion protein system consisting of organophosphorus hydrolase (OPH) and enhanced green fluorescent protein (EGFP) for the rapid and accurate determination of organophosphates (OPs). OPs are well known neurotoxic inhibitors of acetylcholinesterase (AChE). OPH is a hydrolytic enzyme of bacterial origin (Pseudomonas diminuta) capable of catalyzing the hydrolysis of a wide variety of OPs. EGFP is a red-shifted variant of the wild type green fluorescent protein extracted from the jellyfish, Aequorea victoria, whose quantum yield exhibits a pH dependence. In the assay, the OPH domain of the fusion protein catalyzes the hydrolysis of OP substrates, resulting in a slight decrease in the local pH. In response, EGFP undergoes a conformational change in the chromophore, reducing its fluorescence. A construct encoding the fusion protein was developed using recombinant DNA technology, inserted into the pFLAG-MAC expression plasmid, and transformed into DH5α cells where it was expressed and purified utilizing an Anti-FLAG M2 affinity gel column. To assess the relationship between OP substrates and the fusion protein activity, in vitroassays need to be performed. Ultimately, it is believed that a whole cell sensing system is the best suited platform for this unique fusion protein.