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. Carhoxylesterases (hCaE) hydrolyze aliphatic and aromatic esters, and aromatic amides. Carboxylesterase may 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. To this end, clones containing a site-mutated cDNA were prepared and used to stably transform human 293T cells. Transformed 293T cells were grown in a chemostat, and conditions were defined which allow for an optimal time of removal of the culture media for isolation of carboxylesterase. In these studies, enzymatic activity was found to be optimal in cell culture medium at four hours. Enzymatically active carboxylesterase was isolated from the culture media, and the initial steps of enzyme purification were accomplished. Our results indicate that an active recombinant enzyme functions as native enzyme with respect to inhibition by organophosphates and reactivation by oximes.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jul 01, 2002
- Accession Number
- ADA405518
Entities
People
- Kenneth D. Lanclos