Bioscavenging of organophosphate (OP) by human proteins is emerging as a promising medical intervention for prophylaxis and post-exposure treatment against chemical warfare nerve agents. The best-.studied bioscavengers (BSCs) to date, meeting considerable success in pre-clinical research, are human cholinesterases (ChEs). However, ChEs, which are highly efficient in binding and sequestering OPs, are also inactivated by the toxins and therefore administration of large amounts of protein is necessary for full protection, raising the question of the practicality of this approach. The development of improved biocatalysts (BCT) (paraoxonase 1) that can catalytically degrade OPs may address this concern. The proposed effort offers a novel means to biomanufacture recombinant BSCs and BCTs based on the human proteins, butyrylcholinesterase and paraoxonase 1. In collaboration with the other projects participating in the Center, the capacity of these proteins to sequester or hydrolyze OPs will be improved by subjecting their genes to either random in vitro evolution or rational mutagenesis. To that end, we will undertake high-throughput screening of mutant enzyme libraries, in particular to improve the stability and drug-like properties of paraoxonase I. In addition, post-translational glycosylation systems or protein PEGylation procedures will be developed to increase the circulating lifetimes and to eliminate potential antigenicity of proteins produced in non-human, recombinant organisms. The OSU team (Project 6) will utilize the transgenic microalgae, Chlamydomonas reinhardtii, to produce pilot-plant scale quantities of these products for direct recovery from the culture media. In addition, they will optimize this production system for scale-up to commercial production capacity. The primary significance of this project is that it will develop pilot-plant systems for the production of enhanced, second-generation, improved ChE-based BSC and BCT products suitable for human clinical trials.
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