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Cloning, Expression, and Characterization of Ara H 3, a Major Peanut Allergen

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Cloning, Expression, and Characterization of Ara H 3, a Major Peanut Allergen University of New Orleans ScholarWorks@UNO University of New Orleans Theses and Dissertations Dissertations and Theses Fall 12-15-2012 Cloning, Expression, and Characterization of Ara h 3, a Major Peanut Allergen Cathryn E. Garvey University of New Orleans, [email protected] Follow this and additional works at: https://scholarworks.uno.edu/td Part of the Biochemistry Commons, Food Biotechnology Commons, Food Chemistry Commons, Food Microbiology Commons, Molecular Biology Commons, Other Immunology and Infectious Disease Commons, and the Structural Biology Commons Recommended Citation Garvey, Cathryn E., "Cloning, Expression, and Characterization of Ara h 3, a Major Peanut Allergen" (2012). University of New Orleans Theses and Dissertations. 1565. https://scholarworks.uno.edu/td/1565 This Thesis is protected by copyright and/or related rights. It has been brought to you by ScholarWorks@UNO with permission from the rights-holder(s). You are free to use this Thesis in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights- holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/or on the work itself. This Thesis has been accepted for inclusion in University of New Orleans Theses and Dissertations by an authorized administrator of ScholarWorks@UNO. For more information, please contact [email protected]. University of New Orleans ScholarWorks@UNO University of New Orleans Theses and Dissertations Dissertations and Theses 11-5-2012 Cloning, Expression, and Characterization of Ara h 3, a Major Peanut Allergen Cathryn E. Garvey Follow this and additional works at: http://scholarworks.uno.edu/td This Thesis-Restricted is brought to you for free and open access by the Dissertations and Theses at ScholarWorks@UNO. It has been accepted for inclusion in University of New Orleans Theses and Dissertations by an authorized administrator of ScholarWorks@UNO. The author is solely responsible for ensuring compliance with copyright. For more information, please contact [email protected]. Cloning, Expression, and Characterization of Ara h 3, a Major Peanut Allergen A Thesis Submitted to the Graduate Faculty of the University of New Orleans in partial fulfillment of the requirements for the degree of Master of Science in Biological Sciences by Cathryn E. Garvey B.S. University of New Orleans, 2009 December, 2012 Dedication This thesis is dedicated to two of my family members Christopher H. Aldrich and Patrick G. Garvey, who both have severe peanut allergy. ii Acknowledgement I would like to give special thanks to Dr. Soheila Maleki for giving me the opportunity to work and do my research in her laboratory where I learned more about peanuts, molecular biology and protein biochemistry than I could have ever imagined. She was always there to guide me through the process of the demanding and laborious work that research presents and how it can give a person the feeling of defeat. Dr. Maleki was not only a mentor in science and the laboratory but in life experiences. She always presented me with mental challenges and problem solving skills that would prepare me for life not only as a scientist but as a person. I thank her for everything she has given me. I would also like to express my gratitude and thanks to the rest of the Dr. Maleki’s laboratory, Dr. Jacqueline Nesbit, Hsiaopo Cheng, Sanbao Ruan, Jane McBride, and Tysheena Charles. Without them I would have had a difficult time staying motivated and would have been lost at times. Dr. Nesbit, who was a post-doc at the time I started in the laboratory, trained me in a majority of the basic skills I needed to get started on my project. Hsiaopo Cheng always helped me by finding things when I misplaced them and ordered all materials when needed. If I ever had a question about anything, Hsiaopo always had an answer. Sanbao Ruan was extremely helpful during the process and troubleshooting of purifying my protein. Jane McBride taught me a variety of new molecular cloning techniques. Thank you to my good friend Tysheena Charles, who gave me wisdom and advice when needed. She has a gift for teaching and will be leading her own laboratory one day and I wish her luck as she proceeds in her own doctoral studies. Thanks to Dr. Barry Hurlburt. If it were not for his expertise in molecular biology, I would not have excelled and learned so much about how precise and accurate you need to be when dealing DNA. I appreciate all the time and patience he spent with me when it came to designing primers and troubleshooting molecular biology problems I had. He always gave me a feeling a confidence in myself. I learned much from him that I will take with me out into the world of science. I really appreciate the efforts of and attention from Dr. Wendy Schluchter through the entire process of my graduate work at UNO. Without her, none of this would have been possible for me. Dr. Schluchter is irreplaceable both as a professor and a person. She was always there for me to talk to, and when I needed to talk things out regarding research, her door was always open. She is one of the most intelligent and passionate people I have ever encountered and her ability to teach is how I hope to be one day. Talking to her was always amazing and she always presented herself very calm and professional yet easy-going and seemed to have a fun side. I would like to thank Dr. Zhengchang Liu for his aid and fantastic advice, especially as someone who helped me get my graduate studies started. I have learned much from him, as he always challenged iii me in my coursework and any other chance he got. He is a not only a great professor, but is also very passionate in all aspects of science. He amazes me with his vast knowledge and how every time I speak with him, I learn something new. I anticipate the day when I will be able to speak with that kind of knowledge and passion about all areas of science. Dr. Liu is one of UNO’s best professors and assets. I would like to thank the University of New Orleans as a whole for all of the help I received through my long career, starting from undergraduate course work in biology to the end of my graduate research. UNO has changed a great deal since I started as an undergraduate freshman in August 2005, but I am proud to say that I am a UNO graduate not only in May 2009 but again in December 2012. This school has proved itself as a great university that can still challenge its students despite many hardships it has had to endure. Lastly, I would like to thank my family and friends for all the love and support that they have provided to me throughout this time. Without them, I do not know if this experience would have been possible. I thank them for their understanding and patience. My parents, Tom and Terry, who have provided me with an enormous amount of opportunities all my life, have once again gave up some of their time to provide me with the inspiration to reach another one of my goals. I can never thank them enough for everything they have provided to me. I’m especially grateful for my significant other, Jon Leggio. He had words of encouragement and confidence for me every day. iv Table of Contents List of Figures ................................................................................................................................. vi List of Tables ................................................................................................................................ viii Abstract ........................................................................................................................................... ix Introduction ...................................................................................................................................... 1 Materials and Methods ................................................................................................................... 18 Results ............................................................................................................................................ 28 Discussion ...................................................................................................................................... 63 References ...................................................................................................................................... 70 Vita ................................................................................................................................................. 74 v List of Figures Figure Number Figure Title Page Figure 1-1 IgE-Mediated Allergic Response .............................................................................. 4 Figure 1-2 Different Effects of Food Processing ........................................................................ 6 Figure 1-3 Maillard Reaction ...................................................................................................... 7 Figure 1-4 IgE Binding Properties of Roasted vs. Raw Peanuts ................................................. 8 Figure 1-5 Ribbon Diagram of Ara h 3 Monomer .................................................................... 11 Figure 1-6 Modeled Ara h 3 Monomer Depicting N-Glycosylation Sites ................................ 12 Figure 1-7 Ribbon Diagram of Ara h 3 Monomer Depicting N-Glycosylation Sites ............... 12 Figure 1-8 Ara h 3 Monomeric and Hexameric Structures with Original IgE Epitopes
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