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Production of Recombinant Human Butyrylcholinesterase in Nicotiana Benthamiana

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Production of Recombinant Human Butyrylcholinesterase in Nicotiana Benthamiana Production of Recombinant Human Butyrylcholinesterase in Nicotiana benthamiana by Robin L. Hayward A Thesis Presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Environmental Sciences Guelph, Ontario, Canada Robin L. Hayward, September, 2012 ABSTRACT PRODUCTION OF RECOMBINANT HUMAN BUTYRYLCHOLINESTERASE IN NICOTIANA BENTHAMIANA Robin L. Hayward Advisor: University of Guelph, 2012 Professor J. Christopher Hall Nerve agents (NAs) inhibit the essential enzyme acetylcholinesterase. Classified as chemical weapons, NAs are considered a threat to soldiers on the frontlines of warzones. Current treatments can prevent death from NA poisoning, but are not effective in preventing convulsions, seizures, or subsequent brain damage. Butyrylcholinesterase (BChE) binds to NAs, rendering the chemicals harmless to acetylcholinesterase.. Two hundred mg of BChE is the putative prophylactic dose for adult humans, but is difficult to obtain in large quantities from expired human serum. Although recombinant BChE has been expressed in several organisms, the yields are still low. Nicotiana benthamiana is an attractive plant for transient protein production due to its quick growth rate, abundance of tissue, and history of successful recombinant protein production. For this research, N. benthamiana was infiltrated with viral based vectors as well as binary vectors containing the human BChE gene. Multiple assays indicated that binary vector BChE-105-1 + P19 enabled the best expression, producing 26 mg BChE/kg tissue. ACKNOWLEDGEMENTS There are many people, both in the lab and at home, without whom this achievement would not have been possible. Firstly, I would like to thank Dr. J. C. Hall, whose guidance, support and patience is wholeheartedly appreciated. To my committee members Dr. Hung Lee and Dr. Annette Nassuth, I thank you for your advice and direction. To Dr. Michael McLean and Dr. Frey Garabagi I thank you for your interest and expertise. To Linda Veldhuis, Erin Gilbert, and Jessica Rouleau I thank you all for your extensive and valuable technical help, your brainstorming sessions, and your friendship. To John Teat who spent way too many hours infiltrating plants with me, your expertise and patience is appreciated. I would like to thank Haifeng Wang for your advice and superior cloning abilities, and Asma Ziauddin for your enthusiasm and support. I would like to thank Paddy McManus, my office mate and ear lender, and Ashley Meyers, Brittany Grohs, and Joanna Pistilli, whose friendship and advice are appreciated and valued. I would like to thank Ashley Dickson, without whose suggestion I never would have sought after a masters degree, and Gord Furzer who made it happen. Finally, I would like to thank my mom, dad, brother and sister-in-law for endless financial and emotional support. Your belief in my abilities always surpasses my belief in myself. To Amanda, Jay, Tara, and Graham, you have stood by me, encouraged me, and distracted me when necessary. And to Ryan, you may now meet the non-student Robin. Thank you for your support and endless understanding. iii LIST OF ABBREVIATIONS Abbreviation Definition 2-PAM (2-pyridine aldoxime methyl chloride (Pralidoxime) ACh Acetylcholine AChE Acetylcholinesterase AP Alkaline phosphatase AT542 Agrobacterium tumefaciens, strain 542 ATCh Acetylthiocholine BChE Butyrylcholinesterase BHK Baby hamster kidney bp Base pairs BSA Bovine serum albumin BTCh Butyrylthiocholine cDNA Complementary DNA ChAT Choline acetyltransferase CHO Chinese hamster ovary dpi Days post infiltration DTNB Diothio-bis-nitrobenzoic acid ER Endoplasmic reticulum Fab Fragment, antigen binding FBS Fetal bovine serum iv Abbreviation Definition Fuc Fucose FucT Fucosyltransferase GA Tabun GB Sarin GD Soman GF Cyclosarin GOI Gene of interest hBChE Human butyrylcholinesterase HBV Hepatitus B virus IC50 Median inhibition concentration IgA Immunoglobulin A KD Dissociation constant kDa Kilodalton Ki Bimolecular rate constant Km Michaelis constant Kp Phosphorylation constant Ks Reactivation of inhibited enzyme LB Luria bertani media LD50 Median lethal dose mAChR Muscarinic acetylcholine receptor MCS Multiple cloning site mRNA Messenger RNA v Abbreviation Definition NA Nerve agent nAChR Nicotinic acetylcholine receptor nm Nanometre Nos Nopaline synthase O/N Overnight OD Optical density OD600 Optical density at 600 nm OP Organophosphate Osm Osmotin PBST Phosphate-buffered saline with Tween 20 PCR Polymerase chain reaction PTGS Post-transcriptional gene silencing PVX Potato virus X Rbc Ribulose-1,5-bisphosphate carboxylase oxygenase RE Restriction enzyme RER Rough endoplasmic reticulum RNAi RNA interference rpm Revolutions per minute RT-PCR Reverse transcriptase polymerase chain reaction RuBisCO Ribulose-1,5-bisphosphate carboxylase oxygenase scFv Single-chain variable fragment SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis vi Abbreviation Definition T-DNA Transfer DNA TBSV Tomato bushy stunt virus TFF Tangential flow filtration Ti Tumour inducing TMP Transmembrane pressure TMV Tobacco mosaic virus TNB 5-thio-2-nitro-benzoic acid TSP Total soluble protein U Units UTR Untranslated region vir Virulence genes Vmax Maximum velocity Xyl Xylose XylT Xylosyltransferase YEP Yeast extract peptone ΔFX Knock-down N. benthamiana plants with xylose and α-1,3 fucose glycans removed vii TABLE OF CONTENTS Acknowledgements........................................................................................................................iii List of Abbreviations ..................................................................................................................... iv List of Tables .................................................................................................................................. x List of Figures................................................................................................................................ xi 1 General overview and research objectives................................................................................ 1 1.1 Introduction............................................................................................................ 1 1.2 Hypothesis and research objectives ....................................................................... 1 2 Literature review....................................................................................................................... 3 2.1 Cholinergic system of vertebrates.......................................................................... 3 2.1.1 Acetylcholinesterase ....................................................................................... 5 2.1.2 Butyrylcholinesterase...................................................................................... 7 2.2 Organophosphate nerve agents ............................................................................ 10 2.2.1 Cholinesterase inhibition .............................................................................. 12 2.2.2 Symptoms ..................................................................................................... 15 2.3 Treatment of nerve agent poisoning with acetylcholinesterase and butyrylcholinesterase .................................................................................................... 15 2.4 Acetylcholinesterase and butyrylcholinesterase production................................ 18 2.4.1 Mammalian, bacterial and yeast cultures...................................................... 18 2.4.2 Insects ........................................................................................................... 20 2.4.3 Mammals....................................................................................................... 20 2.4.4 Acetylcholinesterase and butyrylcholinesterase in planta............................ 20 2.5 Plant production of therapeutic proteins.............................................................. 24 2.5.1 Agrobacterium tumefaciens .......................................................................... 27 2.5.2 Stable transformation.................................................................................... 27 2.5.3 Transient transformation............................................................................... 29 2.5.4 Glycosylation ................................................................................................ 30 2.6 Enzyme functionality testing with the Ellman assay ........................................... 32 2.7 Restatement of research objectives and hypothesis............................................. 34 3 The production of recombinant human butyrylcholinesterase in Nicotiana benthamiana.................................................................................................................................. 36 3.1 Materials and methods ......................................................................................... 36 3.1.1 Cloning of the butyrylcholinesterase gene into viral-based vectors. ............ 36 3.1.2 Spot infiltrations of Nicotiana benthamiana with ICON genetics modules. 38 3.1.3 Cloning of the butyrylcholinesterase gene into binary vectors..................... 42 3.1.4 Spot infiltrations of Nicotiana benthamiana with binary vectors................. 46 3.1.5 Whole plant infiltration of Nicotiana benthamiana with binary vectors...... 48 3.1.6 Determination of enzyme activity using the Ellman assay........................... 50 3.1.6.1
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