(582e) Expression of Recombinant Human Butyrylcholinesterase in Nicotiana Benthamiana and Its Postproduction In?vitro Glycosylation Modification

Human butyrylcholinesterase (hBChE EC 3.1.1.8) is a 574 amino acid cholinesterase-hydrolyzing enzyme. Organophosphates (OP) are highly toxic inhibitors of the acetylcholine-hydrolyzing enzymes like hBChE. The resulting accumulation of acetylcholine can lead to respiratory collapse and death. Current therapies are based on elevating the serum levels of OP bioscavengers like BChE. The major limitation of this therapy is 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 hBuChE (rhBChE). The development of an effective rhBChE is a pressing national security concern in terms of protecting the nation’s warfighters and civilian population from the threat of attack with OP agents.  

We describe the use of viral amplicon-based gene expression systems based on either Tobacco mosaic virus (TMV) and Cucumber mosaic virus (CMV) to express functional rhBChE in Nicotiana benthamiana using transient agroinfiltration.  Because hBChE is a glycoprotein with nine potential N-glycosylation sites, the glycan structures of the plant-made rhBChE were also characterized. For each expression system, two constructs were made to target the protein to the apoplastic compartment (Apo) of the plant cell or to retain the protein at the endoplasmic reticulum (ER). As expected the variation in subcellular localization resulted in different glycosylation patterns of the recovered butyrylcholinesterase. Retaining the protein at the ER yields a high mannose N-glycans structure, while targeting the protein to the apoplastic compartment yields a complex N-glycan structure. In each of the four different constructs (Apo-TMV, ER-TMV, Apo-CMV and ER-CMV), a 3X FLAG tag is used at N-terminal of the protein for protein identification and purification. Here, we report the expression level of the four different expression systems and the N-glycan structure of the ER retained and apoplast targeted proteins.

Six days post infiltration, the expression level of functional rhBChE using the ER-TRBO, Apo-TRBO, ER-CMVar and Apo-CMVar expression systems are 14.6, 6.6, 0.74 and 2.9 mg/kg FW respectively. The glycan structure of purified ER-retained butyrylcholinesterase reveals almost all the N-glycan having a high mannose structure with insignificant amount of paucimannosidic-type N-glycan. While, the glycan structure of Apo targeted protein reveals a mixture of N-glycans consisting mainly of complex N-glycans (80%), high mannose structure (10%), and paucimannosidic-type N-glycan (10%). We found all nine sites occupied by typical plant glycans. 

It is also known that plants are incapable of sialylating glycoproteins naturally and that sialylation is essential for the normal serum half-life of proteins.  To increase the number of sialic acid residues per rhBChE molecule, we systematically added GlcNAC, galactose and sialic acid to branch-termini of plant N-glycans using multistep enzymatic reactions (i.e., in vitro sialylation). While preliminary results are encouraging, more characterization of ER-retained and apoplast-targeted rBuChE is needed.  Because 30% of all commercial biopharmaceuticals are glycoproteins, these results could make plant-made pharmaceuticals a viable alternative to mammalian or insect expression systems.