Abstract
Although recent innovations in transient plant systems have enabled gram quantities of proteins in 1–2 weeks, very few have been translated into applications due to technical challenges and high downstream processing costs. Here we report high-level production, using a Nicotiana benthamiana/p19 system, of an engineered recombinant human acetylcholinesterase (rAChE) that is highly stable in a minimally processed leaf extract. Lyophylized clarified extracts withstand prolonged storage at 70 °C and, upon reconstitution, can be used in several devices to detect organophosphate (OP) nerve agents and pesticides on surfaces ranging from 0 °C to 50 °C. The recent use of sarin in Syria highlights the urgent need for nerve agent detection and countermeasures necessary for preparedness and emergency responses. Bypassing cumbersome and expensive downstream processes has enabled us to fully exploit the speed, low cost and scalability of transient production systems resulting in the first successful implementation of plant-produced rAChE into a commercial biotechnology product.
Highlights
Of low expression levels, insufficient thermal stability for certain intended applications and/or challenges and costs in downstream processing
We demonstrate that the demanding criteria associated with rapid and cost effective product development have been successfully addressed using an Agrobacterium-mediated transient co-expression system in N. benthamiana (N.b.) plants to produce highly stable rHuAChE in a minimally processed extract form at levels that ensure product effectiveness and economic viability
The initial challenges of low expression and stability of the rHuAChE were overcome in part by co-transfecting three different genes in 6–8 week old N.b. plants: (i) a codon optimized glyco-engineered (D61N and S541N) sequence for the hydrophilic AChE-E4-E6 isoform, in which exon 4 is joined to exon 6, permitting the formation of tetramers (ii) the p19 suppressor of gene silencing from tomato bushy stunt virus for increasing expression levels[16] and (iii) the proline-rich attachment domain (PRAD) of the Colq gene encoding a 17-residue peptide at the N terminus of the AChE collagen tail, which further optimized assembly into tetramers and significantly increased overall expression levels[17]
Summary
Leaf extracts or purified AChE were first diluted in 50 mM phosphate buffer containing 0.5% (w/v) BSA and 10 μ l added to a plate in the presence of 1 mM ATC (Sigma Aldrich) as substrate and 1 mM DTNB. Plant-derived AChE extracts or purified forms using different elution buffers were incubated at ~700 U/ml at 4 °C, RT and 37 °C for 3 weeks to assess stability. For these pot life studies, samples of purified rHuAChE and AChE-containing extract were diluted in 100 mM potassium phosphate pH 7 to achieve the working concentration of 16 U/ml and assessed for stability following incubation in 1 ml aliquots at 40 °C for 0–8 hours. Shelf-life refers to stability of the lyophilized powder, tested immediately after reconstitution, and pot-life refers to enzyme activity over an incubation period after the powder is reconstituted
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