(269f) Agrobacterium Mediated Transient Expression of Butyrylcholinesterase, a Biodefense Agent, in Nicotiana Benthamiana Plants

Organophosphates (OP) are highly toxic inhibitors of the acetylcholine-hydrolyzing enzymes. The resulting accumulation of acetylcholine can lead to respiratory collapse and death. Current therapies are based on elevating the serum levels of OP bioscavengers. The most advanced candidate so far is human butyrylcholinesterase (hBChE). It is a 340 kDa blood protein made in the liver, which is capable of hydrolyzing different choline based esters. The major limitation of this therapy is the high cost, with plasma-derived hBChE costing more than $10,000/treatment. Limitations like cost and availability necessitate an alternative expression platform capable of large scale, low-cost production of a fully active and efficacious recombinant hBChE. The development of an effective recombinant BChE (rBChE) is a pressing national security concern in terms of protecting the nationâ??s warfighters and civilian population from the threat of attack with OP agents. We describe the use of viral amplicon-based gene expression system based on Tobacco mosaic virus (TMV) to express a functional rhBChE in Nicotiana benthamiana plants using a transient agroinfiltration method. A viral gene silencing suppresser (P19) was used to improve the production of the target molecule. In this work, upstream bioprocessing was optimized. The production was monitored at different post infiltration time points using whole plants and harvested leave tissue as a production platform. At each time point, the production level was monitored using SDS-PAGE, western blot and an enzymatic assay (Ellman assay). The maximum production level of functional plant rBChE in whole plants reached 22 0.4 mg/kg fresh weight (FW) 6 days post infiltration, while it reached 41 0.7 mg/kg FW in harvested leaf tissue 12 days post infiltration. The concentration of the agrobacterium harboring the target gene was varied in detached leaves from OD₆₀₀ 0.25 to 1.0, and the optimal OD₆₀₀ was found to be 0.5 giving a yield of 32 0.1 mg/kg FW.