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Developing a 2-Phase Screening System to Assess the in Vitro and in Vivo Stability of Engineered Native Seed Storage Proteins

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Developing a 2-Phase Screening System to Assess the in Vitro and in Vivo Stability of Engineered Native Seed Storage Proteins Developing a 2-Phase Screening System to Assess the in vitro and in vivo Stability of Engineered Native Seed Storage Proteins by Allison F. Jaworski, B.Sc.H A thesis submitted to the Faculty of Graduate and Postdoctoral Affairs in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biology Department of Biology Ottawa-Carleton Institute of Biology Carleton University Ottawa, Ontario © 2015, Allison F. Jaworski Abstract. Seed storage proteins are the nitrogen and sulfur reserves for the germinating embryo and comprise the majority of the protein content of a seed (Shewry et al, 1995; Tandang-Silvas et al, 2010). The 11S globulins are the principal seed storage proteins in members of the legume and mustard families. They are targets for protein engineering studies attempting to alter the physicochemical properties (e.g. emulsification and gelling) and nutritional value, with respect to amino acid content, of their seed protein extracts (Muntz et al, 1998; Tandang-Silvas et al, 2011). The factor that has limited the success of these studies to date is insufficient accumulation of the engineered variants in vivo due to their reduced stability, compared to the native protein. To address this issue, this thesis describes the development of a 2-phase screening system that facilitates protein engineering of native 11S globulins by assessing the ability of stable site-directed variants to accumulate in seeds. Phase I employs E. coli as an expression system to assess the thermodynamic stability of the engineered seed storage proteins in vitro. Methionine-enriched 11S globulins with stability parameters similar to the wild-type protein are then expressed in A. thaliana in phase II, the in vivo component of the screen, to assess the ability of the engineered protein to accumulate in plant seeds. To complement the novel 2-phase screening system, a comprehensive survey of 67-methionine-enriched variants of a model A. thaliana 11S globulin was completed, employing fluorescence-monitored guanidine hydrochloride denaturation to identify regions of the protein that are amenable to the introduction of methionine residues. This survey demonstrates that methionine can be introduced into 11S globulins but single locations cannot be treated as sinks for the introduction of multiple, sequential methionine residues. The use of HotSpot Wizard in conjunction with available structural data is a successful strategy for short-listing residues as targets for substitution to methionine. ii This thesis developed tools, a 2-phase screen and an information platform identifying regions within the 11S globulin structure that are accepting of methionine introduction, to facilitate subsequent protein engineering studies attempting to alter the physicochemical or nutritional properties of seed proteins. iii Statement of Contributions. My contributions to the research described in this thesis include: 1. The development of the research questions for each data chapter in addition to the overarching objective of this thesis to create a 2-phase screening system to use in protein engineering studies focusing on improving the physicochemical and nutritive values of seed storage proteins in collaboration with Dr. S.M. Aitken 2. The responsibility of developing and establishing the experimental design of each data chapter 3. The collection and analysis of data for each of the three data chapters 4. The writing and preparation of the published manuscript (Chapter 2) in collaboration with Dr. S.M. Aitken. I formally acknowledge the contributions of the co-author, Dr. S.M. Aitken, of the manuscripts that comprise the research chapters of my thesis. In a collaborative effort, my supervisor, Dr. S.M. Aitken, contributed her expertise to the development of the research questions and experimental design in addition to the preparation of manuscripts for the research chapters of my thesis, and providing insights and guidance throughout the writing of my thesis. iv Acknowledgements. I want to express my sincere gratitude to Dr. Susan Aitken for her mentorship, encouragement, guidance and for giving me the opportunity to come into her lab and learn from her and all her talented students. These past six years have not only helped mold my scientific mind but have shaped me into the person I am today. I also want to thank her so much for always encouraging me to strive past any preconceived notions that I (and others) have had pertaining to who I am and what I am capable of in order to surpass those notions and show myself what I really can do – which has surprised me, and made me proud, to say the very least. This has helped shape me into the scientist and person I am now and I am grateful that she has helped me to stretch in her lab to reach for the stars so that I now know I can. I would also like to thank the numerous members of the Carleton Biology Department that have given me invaluable advice, feedback and support throughout my graduate studies including my committee members Dr. Owen Rowland and Dr. John Vierula, and Dr. Ashkan Golshani, Dr. Amanda MacFarlane, Dr. Andrew Simmons, Dr. Suzanne Patterson and Dr. Alex Wong. Finally I want to thank my family and friends for supporting me through the ups and downs of grad student life. It could not have been easy – the meanest rollercoaster in the biggest amusement park couldn’t compare – but you all helped keep me grounded and encouraged me to strive to be the very best I could be. Thank you so much for always being there. v Dedication. To my family Mom, Dad, Paul, Nicole, Val, Gregg and Georgia To my friends Alex, Mohsen, Duale, Eddy, Bill, Bahram, Matt, Aimee, Dan, Kristina, Dominique, Katie, Magda, Maryam, Mary and Darlene for always believing I’m superwoman and can do anything I put my mind to. vi Table of Contents. Abstract. ............................. ii Statement of Contributions. ..................... iv Acknowledgements. ........................ v Dedication. ............................ vi Abbreviations. ......................... xiii List of Tables ........................... x List of Figures ........................... xi Chapter 1: Introduction. ....................1 1.1. Methionine in Grain Legumes. ................. 1 1.2. Current Approaches for Addressing Amino Acid Deficiencies in Crops. ......................... 3 1.3. Seed Proteins. ........................ 8 1.3.1. Seed Storage Proteins ..................10 1.3.1.1. 2S Albumins. ..................12 1.3.1.2. Prolamines and Glutelins. .............14 1.3.1.3. Globulins. ....................14 1.3.1.3.1. 7S Globulins. ...............18 1.3.1.3.2. 11S Globulins. ..............18 1.4. Developing a Strategy to Sequester more Methionine in the Seed. 20 1.4.1. Developing a 2-Phase Screening System to Facilitate Future Protein Engineering Studies on Native Seed Storage Proteins. ....21 Chapter 2: Expression and Characterization of the Arabidopsis thaliana 11S globulin family. ................. 23 2.1. Abstract. ......................... 23 2.2. Introduction. ....................... 23 vii 2.3. Materials and Methods. ....................27 2.3.1. Reagents. .......................27 2.3.2. Amplification ofA. thaliana Procruciferin Coding Sequences. 27 2.3.3. Optimization of Expression and Purification Conditions for the Affinity-Tagged Procruciferins. ...............27 2.3.4. Urea Denaturation of His-proAtCRU Proteins. ...... 29 2.3.5. Differential Scanning Fluorimetry (DSF) of His-proAtCRU Proteins. ........................ 30 2.4. Results and Discussion. ................... 30 2.4.1. Optimization of Expression and Purification Conditions. .. 30 2.4.2. Fluorescence Properties and Relative Stability of the A. thaliana Procruciferins. .......................37 2.5. Concluding Remarks. .................... 42 Chapter 3. Developing an in vivo Expression System to Assess Accumulation of Engineered 11S Globulins. .......... 43 3.1. Abstract. ......................... 43 3.2. Introduction. ....................... 44 3.3. Methods and Materials ................... 46 3.3.1. Reagents. ...................... 46 3.3.2. Constructing the Plant Vectors. ..............47 3.3.2.1. Promoter-Reporter Constructs. ...........47 3.3.2.2. Cruciferin-HA tag Constructs. ...........47 3.3.3. Transformation of Agrobacterium. ........... 50 3.3.4. Floral dip Transformation of A. thaliana. ......... 50 3.3.5. Selecting Transgenic A. thaliana. ............ 51 3.3.6. GUS Staining of Transgenic A. thaliana Siliques and Seeds. .. 52 viii 3.3.7. Purification of His-proAtCRU3-HA tag fromEscherichia coli. 52 3.3.8. Seed Protein Extraction from A. thaliana. ........ 53 3.3.9. Immunoblot. .................... 54 3.4. Results and Discussion. ....................54 Chapter 4: Surveying Structural Components of 11S Globulins for Locations Amenable to the Introduction of Methionine Residues . 61 4.1 Abstract. ..........................61 4.2 Introduction. ....................... 62 4.3 Methods and Materials. ....................67 4.3.1. Reagents. .......................67 4.3.2. Designing and Constructing Methionine-Enriched proAtCRU3 Variants. .....................67 4.3.3. Expression and Purification of the Methionine-Enriched His-proAtCRU3 Variants. .................. 68 4.3.4. Guanidine Hydrochloride (GdnHCl) Denaturation of His-proAtCRU3 Methionine-Enriched Variants. ....... 69 4.4 Results. .......................... 70 4.4.1. Variants Within the Cupin Domains. ........... 83 4.4.2. Variants Within the Hypervariable Regions. ........ 89 4.5 Discussion. .......................
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