Transgenic plants as a source for the bioscavenging enzyme, human butyrylcholinesterase Brian C. Geyer 1 , Latha Kannan 1 , Irene Cherni 1 , Ryan R. Woods 1 , Hermona Soreq 2 and Tsafrir S. Mor 1,* 1 School of Life Sciences and The Biodesign Institute, Arizona State University, Tempe, AZ, USA 2 Department of Biological Chemistry, The Hebrew University of Jerusalem, Jerusalem, Israel Received 15 September 2009; revised 15 September 2009; accepted 3 February 2010. *Correspondence (Tel 480 727 7405; fax 480 965 6899; email tsafrir.mor@asu.edu) Accession numbers: pBCHE GQ903330 Keywords: organophosphorous compounds, nerve agents, pesticides, codon usage, transgenic plants, enzyme therapy. Summary Organophosphorous pesticides and nerve agents inhibit the enzyme acetylcholines- terase at neuronal synapses and in neuromuscular junctions. The resulting accumula- tion of acetylcholine overwhelms regulatory mechanisms, potentially leading to seizures and death from respiratory collapse. While current therapies are only capable of reducing mortality, elevation of the serum levels of the related enzyme butyrylcholinesterase (BChE) by application of the purified protein as a bioscavenger of organophosphorous compounds is effective in preventing all symptoms associated with poisoning by these toxins. However, BChE therapy requires large quantities of enzyme that can easily overwhelm current sources. Here, we report genetic optimi- zation, cloning and high-level expression of human BChE in plants. Plant-derived BChE is shown to be biochemically similar to human plasma-derived BChE in terms of catalytic activity and inhibitor binding. We further demonstrate the ability of the plant-derived bioscavenger to protect animals against an organophosphorous pesticide challenge. Introduction Oragnophosphorous (OP) compounds are highly toxic inhibitors of the acetylcholine-hydrolyzing enzymes acetyl- cholinesterase (AChE) and butyrylcholinesterase (BChE). Although first explored as insecticides, the extreme toxicity of OPs towards mammals prompted their development as chemical warfare agents, and the first military grade OP nerve agents, tabun, sarin and soman, were synthesized in Nazi Germany immediately prior to and during World War II. The cold war era saw the unfortunate proliferation of the technology and the development of yet more toxic compounds such as VX, Russian-VX and cyclosarin (Green- field et al., 2002; Lee, 2003; Johnson et al., 2009). In fact, nerve agents are relatively easy to produce, store and weaponize, and their use by terrorists and rogue govern- ments (notoriously exemplified by the Tokyo subway sarin attack by Aum Shinrikyo in 1995) pose a major threat to civilians and military personnel (Johnson et al., 2009). In addition, the widespread and sometimes irresponsible use of OP pesticides increases the chance of environmental and occupational exposure of individuals and communities (Ray and Richards, 2001; Catano et al., 2008; Jintana et al., 2009). Such concerns over exposure from overuse and misuse of OP pesticides and nerve agents have led to detailed studies of how such agents work and how to prevent or ameliorate their effects. However, developing safe and effective pro- phylactic treatments was met with considerable complica- tions. For over a decade, application of cholinesterases, especially BChE, acting as scavengers of OPs has been explored as a promising medical intervention for prophylaxis and post-exposure treatment against nerve agents. In the near term, outdated human plasma can be a first genera- tion source of BChE for human clinical trials to validate its safety (Saxena et al., 2006). In the longer term, a cost- effective, sustainable supply of cholinesterases from an alternative source must be identified to establish and main- tain a strategic reserve of the bioscavengers as they are needed in stoichiometric rather than catalytic quantities. ª 2010 The Authors Plant Biotechnology Journal ª 2010 Society for Experimental Biology and Blackwell Publishing Ltd 873 Plant Biotechnology Journal (2010) 8, pp. 873–886 doi: 10.1111/j.1467-7652.2010.00515.x