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The Characterization of a Recombinant Virophage Integrase

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The Characterization of a Recombinant Virophage Integrase University of Texas at El Paso DigitalCommons@UTEP Open Access Theses & Dissertations 2016-01-01 The hC aracterization of A Recombinant Virophage Integrase Martin Christopher Chacon University of Texas at El Paso, [email protected] Follow this and additional works at: https://digitalcommons.utep.edu/open_etd Part of the Biochemistry Commons, Chemistry Commons, and the Virology Commons Recommended Citation Chacon, Martin Christopher, "The hC aracterization of A Recombinant Virophage Integrase" (2016). Open Access Theses & Dissertations. 621. https://digitalcommons.utep.edu/open_etd/621 This is brought to you for free and open access by DigitalCommons@UTEP. It has been accepted for inclusion in Open Access Theses & Dissertations by an authorized administrator of DigitalCommons@UTEP. For more information, please contact [email protected]. THE CHARACTERIZATION OF A RECOMBINANT VIROPHAGE INTEGRASE MARTIN CHRISTOPHER CHACON Master’s Program in Chemistry APPROVED: Chuan Xiao, Ph.D., Chair Mahesh Narayan, Ph.D. Manuel Llano, Ph.D. Charles Ambler, Ph.D. Dean of the Graduate School Copyright © by Martin Chacon 2016 Dedication Dedicated to my family. Thank you for all of your support on my journey. THE CHARACTERIZATION OF A RECOMBINANT VIROPHAGE INTEGRASE by MARTIN CHRISTOPHER CHACON, B.S. Biochemistry THESIS Presented to the Faculty of the Graduate School of The University of Texas at El Paso in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE Department of Chemistry THE UNIVERSITY OF TEXAS AT EL PASO August 2016 Acknowledgements This research was conducted in the Department of Chemistry at UTEP. I would like to thank my mentor, Dr. Chuan (River) Xiao (Assistant Professor, Department of Chemistry at UTEP) for his mentorship. Dr. Xiao inspired me to want to join his lab after a semester of his biochemistry course. Luckily he took me in, even though I only got a B in his course. Under his mentorship, I strove for greatness and was able to obtain an A for his biochemistry 2 course. This is just one very small example of how he has pushed me throughout my research career. He is the reason I completed this thesis and why I am continuing my studies as doctoral candidate at UTEP. Dr. Xiao has spent a large amount of time to help bring this thesis to its final state. I would also like to thank Dr. Mahesh Narayan (Assistant Chair and Chemistry Graduate Advisor, Department of Chemistry at UTEP) for allowing me to use his instruments, and for his advice during my Master’s candidacy. I would like to thank Dr. Manuel Llano (Associate Professor, Department of Biology at UTEP), for his insight and guidance for my project. I would like to thank Dr. Xiao, Dr. Narayan, and Dr. Llano as my thesis committee members. They were essential in my studies on my project. I would like Duer Bolotaulo. He was my mentor for laboratory techniques when I first started working in Dr. Xiao’s lab. Duer always put up with my questions and helped guide me to find the answers. He was a large component of this project as he is the one who started it. I would like to thank Dr. Supriyo Ray, he has taken the time and energy to teach me new techniques to complete my thesis and project. He is also a very huge component of this work. I also want to thank my former and current lab members. They have all taught me in many ways, even if they do not realize it. I want to thank Dr. Jianjun Sun (Assistant Professor, Department of Biology at UTEP) for providing the pCold-TF vector. I would like to thank Dr. Matthias G. Fischer at Max Planck Institute for Medical Research, Heidelberg, Germany for providing many materials and data for v this project. I would like to thank Dr. Hugues Oullet (Assistant Professor, Department of Biology at UTEP) and his graduate student Uriel Ortega-Rodriguez for verifying the primary sequence of the expressed recombinant protein, MV02, using mass spectrometry. I would like to thank the BUILDing scholars program for funding some of the instruments that were used for this project. I would like to thank the Border Biomedical Research Center (BBRC) for providing many instruments and services that were essential for this research. I would like to thank the SMARTS program for providing me funding that helped pay my tuition, research, and travel to research conferences. I would like to thank the department of Chemistry at UTEP for providing me a teaching assistant position and for their help during my studies. I would like to thank the UTEP Graduate School for providing funding in the means of travel grants and the Dodson Research grant. Also I would like to thank them for their free workshops that helped me along the way as a graduate student. I would especially like to thank Tiffany Robles, my former undergraduate student. She helped move my project forward as I prepared this thesis. I would like to thank Yuejiao Xian, a former foreign exchange student, now a current lab mate as a graduate student. She has helped me learn so much, and encouraged me along the way. Yuejiao has helped design some of the figures that are presented in this thesis. Thank you all for helping me in all the ways you did. Without all of you, my research would have been more difficult. vi Abstract Virophages are satellite-like dsDNA viruses that parasitize giant viruses of the family Mimiviridae. Mavirus is the second virophage discovered that associates with its host virus Cafeteria roenbergensis Virus (CroV). When co-infecting their common host cell Cafeteria roenbergensis, a marine zooplankton that is widely spread throughout the oceans, mavirus will inhibit CroV’s replication. In addition, mavirus was shown to share high similarities to the Maverick/Polinton eukaryotic DNA transposons. A coding sequence in mavirus genome (MV02) reveals high homology to retroviral integrases such as those found in HIVs. The putative integrase MV02 is predicted to integrate mavirus DNA into the host genome. In order to characterize MV02 and prove it is indeed an integrase, large amounts of protein need to be obtained and three functional aspects must be studied that include: DNA binding, DNA processing, and DNA strand transfer. In this study, large amount of recombinant MV02 has been expressed and purified to high homogeneity. The recombinant MV02 has been tested for its DNA binding using chromatographic techniques and gel shift assays. These experiments build the foundation for future structural studies as well as further functional analysis such as testing DNA processing and strand transfer capabilities of recombinant MV02. Proving MV02 is an integrase will not only reveal an aspect of the virophage infection process, but will also shed light on the evolutionary relationship between the mavirus and the Maverick/Polinton transposons. Due to high similarity between MV02 and retroviral integrase and limited structural and functional knowledge of the latter, new information obtained by studying mavirus integrase will shed new insights for drug development against AIDS. vii Table of Contents Acknowledgements ..........................................................................................................................v Abstract ......................................................................................................................................... vii Table of Contents ......................................................................................................................... viii List of Tables ...................................................................................................................................x List of Figures ................................................................................................................................ xi Chapter 1: Introduction ....................................................................................................................1 1.1. Satellite Viruses vs Virophages .......................................................................................1 1.2. Importance of Host Giant Virus Virion Factories for Virophage Replication.................2 1.3. Integration of Mavirus DNA into the Host Genome .......................................................5 1.4. Project Goal .....................................................................................................................6 Chapter 2: Materials and Methods ...................................................................................................8 2.1. Recombinant MV02 Design and Expression ...................................................................8 2.1.1. MV02 Expression Construct ................................................................................8 2.1.1. Recombinant TF-MV02 Expression ....................................................................8 2.2. Purification of Recombinant TF-MV02...........................................................................9 2.2.1 Affinity Chromatography......................................................................................9 2.2.2. Ion-Exchange Chromatography ...........................................................................9 2.2.3. Size-Exclusion Chromatography .......................................................................10 2.3. Thrombin Digest Optimization and Purification of Recombinant MV02 .....................10 2.4. Protein-DNA Interaction Studies of Recombinant TF-MV02 .......................................11 2.4.1. Heparin Chromatography on Recombinant Purified TF-MV02
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