EXPRESSION AND Purification of a Potential antidote for Organophosphate Warfare Agents

Abstract : Serine-dependent carboxylesterases (E.C.3.1.1.1) are found in a variety of tissues with high activity detected in the human liver. Carboxylesterases (CaE) hydrolyze aliphatic and aromatic esters, and aromatic amides; and play an important role in the detoxification of xenobiotic chemicals that contain organophosphate (OP) compounds. Thus, an injectable form of human hCaE should prove to be a valuable antidote for protecting soldiers from these chemical agents. The goals of this project are to over express a functional human liver hCaE from a recombinant cDNA in a human cell line, and isolate and purity the recombinant protein. To accomplish these goals, the cDNA encoding hCaE was altered in order to convert it to a secretory form. Expression of the site-mutated cDNA in cell culture resulted in the secretion of an active hCaE into the growth medium. Thus, the secreted hCaE enzyme will be concentrated and purified using hydrophobic interaction chromatography, Cibacron blue affinity chromatography, and isoelectric focusing chromatography. The long-term goal of the project is to isolate quantities sufficient for crystallization with organophosphate agents and to evaluate its use for protection by enzymatic detoxification of organophosphate nerve agents in an animal model.