Human butyrylcholinesterase (BChE), historically referred to plasma or serum cholinesterase, has been found in nearly every tissue in humans. Although its physiological role is unknown, the enzyme’s substrate and inhibitor selectivities largely overlap with the more thoroughly characterized acetylcholinesterase (AChE). BChE can scavenge and therefore provide protection against administered or inhaled poisons that target AChE and similar physiological targets. In animal studies, treatment with BChE has provided protection from exposure of up to 5 × LD50
of chemical nerve agents that target AChE [1
], and in 2010, FDA approval was given to develop the BChE as a therapeutic drug for prophylactic treatment against nerve agent exposure [2
]. In addition, BChE variants are currently being pursued by various laboratories attempting to generate novel enzymes with enhanced ability to hydrolyze organophosphate ester (OP)-based nerve agents and other toxic compounds, including cocaine [3
]. As the diversity and application of novel BChE variants develops, so does the need for a robust purification protocol independent of a given variant’s primary structure. Currently, commonly used purification methods rely on BChE affinity for procainamide, a small molecule that binds the enzyme’s substrate binding site [9
]. Some variants however, including the previously characterized G117H/E197Q BChE [10
], have little affinity for procainamide, and therefore are not efficiently purified using a procainamide column (Lockridge, personal communication). In light of the above noted efforts to develop novel BChE variants, there is a need for a more robust purification method that is insensitive to changes within the enzymes primary structure. Affinity tags are a logical solution.
Addition of affinity tags to BChE is potentially complicated by posttranslational processing of the recombinant protein. The BChE gene encodes an N-terminal sequence targeting the enzyme for secretion from mammalian cells. The N-terminal sequence of BChE is post-translationally cleaved to generate a mature enzyme, making N-terminal affinity tags problematic. Similarly, Blong et al. (1997) documented a significant amount of post-translational proteolysis of the C-terminus during recombinant expression. Preliminary attempts to utilize C-terminal tags provided only 30% recovery of enzymatic functional activity during protein purification with affinity columns (unpublished results). Studies have shown that the C-terminus of BChE is involved in tetramerization and is not essential for catalysis [11
]. As many as 50 amino acids can be removed from the C-terminus of wild-type BChE in the cloning stage without large changes in the observed kinetic parameters after expression [11
]. One study reported the successful use of a C-terminal His6
-tag on truncated BChE enzyme for metal-chelate interaction chromatography (MIC)-based purification [14
]. However, the MIC step was applied after significant purification was achieved by ammonium sulfate precipitation and procainamide affinity chromatography, and the recovery efficiency was not reported. To date, no other His-tagged monomeric BChE variants have been reported in the literature. In the current manuscript, a truncated His6
-tagged construct generating W541H6
Δ BChE variants was used for expression of wild-type and two previously reported BChE variants: G117H and G117H/E197Q BChE [10
]. The latter enzyme is known to have poor affinity for conventional procainamide resins. The enzymes were purified via MIC methods and characterized for functional hydrolase activity. Enzymes (wild-type and G117H/E197Q) were further characterized for the rate of inhibition with the OP echothiophate (ETP), the rate of spontaneous reactivation (G117H), and the rate of reactivation with pyridine-2-aldoxime methiodide (2-PAM) or MMB4 after inhibition by a nerve agent model compound (wild-type).