435051 Recovery and Purification of Recombinant Butyrylcholinesterase from Nicotiana Benthamiana Plants

Wednesday, November 11, 2015: 8:48 AM
150D/E (Salt Palace Convention Center)
Salem Alkanaimsh1, Kalimuthu Karuppanan1, Somen Nandi2, Raymond Rodriguez2 and Karen A McDonald1, (1)Chemical Engineering and Materials Science, University of California, Davis, Davis, CA, (2)Molecular & Cellular Biology, University of California, Davis, Davis, CA

Recovery and Purification of Recombinant Butyrylcholinesterase from Nicotiana benthamiana Plants

Salem Alkanaimsh, Kalimuthu Karuppanan and Karen A. McDonald

Chemical Engineering and Materials Science, University of California, Davis, CA, 95616

Human butyrylcholinesterase (hBuChE EC is a 574 amino acid cholinesterase-hydrolyzing enzyme. Organophosphates (OP) are highly toxic inhibitors of the acetylcholine-hydrolyzing enzymes like acetylcholinesterase. 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 BuChE. The major limitation of this therapy is high cost, with plasma-derived hBuChE 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 (rhBuChE). The development of an effective rhBuChE 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. Therapeutic proteins (e.g. hBuChE) can be produced in plant leaves such as Nicotiana benthamiana using transient agroinfiltration. Nicotiana benthamiana represents a valuable biopharmaceutical protein expression system due to its scalability, ability to glycosylate proteins and elimination of risk of viral infection. We have a developed a viral expression system based on Tobacco mosaic Virus (TMV) that express a FLAG tagged rhBuChE at a level of 10 µg/g fresh weight. Downstream processing is a major challenge of Nicotiana benthamiana based where it represents 80% of the overall production costs ADDIN EN.CITE <EndNote><Cite><Author>Wilken</Author><Year>2012</Year><RecNum>170</RecNum><DisplayText><style R.</author><author>Nikolov, Zivko L.</author></authors></contributors><titles><title>Recovery and purification of plant-made recombinant proteins</title><secondary-title>Biotechnology Advances</secondary-title></titles><periodical><full-title>Biotechnology Advances</full-title></periodical><pages>419-433</pages><volume>30</volume><number>2</number><keywords><keyword>Recombinant protein</keyword><keyword>Purification</keyword><keyword>Downstream processing</keyword><keyword>Transgenic plants</keyword><keyword>Protein extraction</keyword><keyword>Bioseparations</keyword><keyword>Process economics</keyword><keyword>Extraction</keyword><keyword>Chromatography</keyword></keywords><dates><year>2012</year><pub-dates><date>3//</date></pub-dates></dates><isbn>0734-9750</isbn><urls><related-urls><url>http://www.sciencedirect.com/science/article/pii/S0734975011001303</url></related-urls></urls><electronic-resource-num>http://dx.doi.org/10.1016/j.biotechadv.2011.07.020</electronic-resource-num><access-date>2012/4//</access-date></record></Cite></EndNote>1. Thus, development of an effective downstream process for purifying rhBuChE is important. Use of an anti-FLAG affinity resin to capture rhBuChE gives only a 20% yield recovery. Procainamide resin which tightly binds to cholinesterases can be used to purify the protein and to enhance the yield percentage. However prior to this chromatography step, different pretreatment strategies were employed such as pH reduction, ammonium sulfate fractionation, polyethylene glycol (PEG) fractionation or two phase aqueous separation of plant extracts to remove plant pigments and native plant protein (e.g. Rubisco).

 ADDIN EN.REFLIST 1.            Wilken, L.R. & Nikolov, Z.L. Recovery and purification of plant-made recombinant proteins. Biotechnology Advances 30, 419-433 (2012).

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