Expression of Recombinant Products with Butyrylcholinesterase (BChE) Activity in Pichia pastoris

Background/Project Significance: Butyrylcholinesterase (BChE) is a serine hydrolase used as a prophylactic countermeasure against organophosphate nerve agents, like V-series gases and sarin. BChE binds and neutralizes nerve agents in the bloodstream before the toxins can inhibit native cholinesterase function in the nervous system. BChE is currently the only therapeutic agent effective in protecting humans against the entire spectrum of organophosphate nerve agents. Procurement of measurable quantities of this reagent for human treatment remains challenging mainly due to its complex biophysical characteristics: native circulating BChE is a ~270 kD tetramer of heavily glycosylated subunits bearing N-linked glycans at each of nine different sites. Current recommendations for use of BChE as a prophylactic measure against nerve agents require doses of ~400 mg per person (provided in 8-16 mL intravenous bolus at 25 mg/mL) with neutralizing activity of 621 U/mg and a circulating half-life of 10 days. While BChEs functional activity is independent of both BChE glycosylation and tetramerization, longevity is largely determined by glycosylation, specifically sialylation, and may be improved with increased tetramerization. To best achieve this target product profile (TPP), BChE typically is isolated from human plasma Cohn fraction IV. Given the overall protein complexity of human plasma and starting purity of BChE within it, the isolation process requires expensive affinity resins like procainamide or huprine and requires an excess of 400 kg starting material to yield only ~8g of pharmaceutical-grade BChE product. Given these low product yields and the associated costs, prophylactic doses of BChE rarely are provided beyond those given to at-risk active military personnel.