Abstract
To optimize the expression, extraction and purification of plant-derived tetrameric recombinant human butyrylcholinesterase (prBChE), we describe the development and use of plant viral amplicon-based gene expression system; Tobacco Mosaic Virus (TMV) RNA-based overexpression vector (TRBO) to express enzymatically active FLAG-tagged plant made recombinant butyrylcholinesterase (rBChE) in Nicotiana benthamiana leaves using transient agroinfiltration. Two gene expression cassettes were designed to express the recombinant protein in either the ER or to the apoplastic compartment. Leaf homogenization was used to isolate ER-retained recombinant butyrylcholinesterase (prBChE-ER) while apoplast-targeted rBChE was isolated by either leaf homogenization (prBChE) or vacuum-extraction of apoplastic wash fluid (prBChE-AWF). rBChE from apoplast wash fluid had a higher specific activity but lower enzyme yield than leaf homogenate. To optimize the isolation and purification of total recombinant protein from leaf homogenates, an acidic extraction buffer was used. The acidic extraction buffer yielded >95% enzymatically active tetrameric rBChE as verified by Coomassie stained and native gel electrophoresis. Furthermore, when compared to human butyrylcholinesterase, the prBChE was found to be similar in terms of tetramerization and enzyme kinetics. The N-linked glycan profile of purified prBChE-ER was found to be mostly high mannose structures while the N-linked glycans on prBChE-AWF were primarily complex. The glycan profile of the prBChE leaf homogenates showed a mixture of high mannose, complex and paucimannose type N-glycans. These findings demonstrate the ability of plants to produce rBChE that is enzymatically active and whose oligomeric state is comparable to mammalian butyrylcholinesterase. The process of plant made rBChE tetramerization and strategies for improving its pharmacokinetics properties are also discussed.
Highlights
Organophosphate (OP) nerve agents such as Sarin (Pohanka et al, 2013) have been used in recent history against civilian populations in Japan (Seto et al, 1999) and in Charbonneau and Nichols (2013)
The results of this study indicate that plant-derived tetrameric recombinant human butyrylcholinesterase (prBChE), extracted from whole leaf homogenates was similar to the hBChE and equine butyrylcholinesterase (eqBChE) controls in terms of physiochemical properties, tetramerization and kinetic parameters
Total protein from leaf homogenates were extracted with tris buffered saline (TBS)-1 and the level of active prBChE-endoplasmic reticulum (ER) was found to be approximately the same as the prBChE enzyme (Figure 2)
Summary
Organophosphate (OP) nerve agents such as Sarin (Pohanka et al, 2013) have been used in recent history against civilian populations in Japan (Seto et al, 1999) and in Charbonneau and Nichols (2013) Their acute toxicity is due to an irreversible inhibition of human acetylcholinesterase (hAChE; Moshiri et al, 2012), which leads to accumulation of acetylcholine in the synaptic cleft followed by overstimulation of cholinergic receptors and death if left untreated (Trovaslet-Leroy et al, 2011). Because the concentration of hBChE in the blood is very low, extracting and purifying gram or kilogram quantities of hBChE from blood is impractical (Browne et al, 1998) This has led researchers to explore heterologous expression systems to produce functional recombinant hBChE in amounts that are affordable and practical for protecting civilian and military personnel from OP nerve agents. These systems are capable of producing functional rBChE, their drawbacks include high fermentation cost, slow cell growth rates, risk of viral infection of mammalian cell cultures and long lag time between gene transfer and lactation for transgenic animals (Houdebine, 2009; Thomas et al, 2011)
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