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Production of Double-Layered Rotavirus-Like Particles in A Engineering yeast strains for the expression of South African G9P[6] rotavirus VP2 and VP6 structural proteins By Mohau Steven Makatsa Dissertation submitted in fulfilment of the requirements for the degree Magister Scientiae in the Department of Microbial, Biochemical and Food Biotechnology Faculty of Natural and Agricultural Sciences University of the Free State Bloemfontein South Africa Supervisor: Dr. Hester G. O’Neill Co-supervisor: Prof. Jacobus Albertyn February 2015 I can do all things through Christ who strengthens me. - Philippians 4:13 Problems are not stop signs, they are guidelines. - Robert H. Schuller Our greatest weakness lies in giving up. The most certain way to succeed is always to try just one more time. - Thomas A. Edison The secret of getting ahead is getting started. - Mark Twain ii To my daughter Reitumetse Makatsa, you are the greatest blessing in my life iii Declaration “I Mohau Steven Makatsa declare that the dissertation hereby submitted by me for the Magister Scientiae degree at University of the Free State is my own independent work and has not previously been submitted by me at another university/faculty. I further more cede copyright of the dissertation in favour of University of the Free State” Signature: Mohau Steven Makatsa Department of Microbial, Biochemical and Food Biotechnology, University of the Free State, South Africa. iv Acknowledgements I would like to express my sincerest gratitude towards the following people and institutions: God for the gift of life Dr. Hester G O’Neill for providing me with this opportunity to pursue my postgraduate study under her supervision. Thank you for your guidance, encouragements and professional and personal support. Your love and passion for science inspires me Prof. Jacobus Albertyn for all your useful suggestions during this work, you inspired me and challenged me to think critically as a scientist Prof. J Görgens, Stellenbosch University for providing us with Pichia pastoris/Hansenula polymopha codon-optimized open reading frames encoding VP2 and VP6 rotavirus structural proteins Prof. A. Van Dijk, North-West University for providing us with VP6 rotavirus protein inserted in pCOLD vector Aliza Naude, an M.Sc graduate from the North-West University for conducting the previous work on expression of rotavirus VP2 and VP6 structural proteins in yeast. Valerie Oberhardt, a graduate student from the Ruprecht-Karls-Universität Heidelberg, Germany, and DAAD/RISE (German Academic Exchange Service) intern for construction of pcDNA3.1_VP6 (His-tag) Katlego S Mthethwa, a post-graduate student in the Department of Microbial, Biochemical and Food Biotechnology, University of the Free State for providing us with Baeyer–Villiger monooxygynase (BVMO) protein My colleagues at the Department of Microbial, Biochemical and Food Biotechnology. Special thanks to colleagues in Molecular Virology, Clinical Biochemistry and Molecular Biology laboratories National Research Foundation (NRF) and Poliomyelitis Research Foundation (PRF) for financial support My family for all the support and encouragement throughout my studies v Contents Page Declaration ..................................................................................................................... iv Acknowledgements .......................................................................................................... v List of Figures .................................................................................................................. ix List of Tables .................................................................................................................. xii Abbreviations .................................................................................................................xvi Conference presentations ............................................................................................. xviii CHAPTER 1 Introduction to the study .................................................................................................. 1 1.1 Background ............................................................................................................................. 1 1.2 Problem identification ............................................................................................................ 2 1.3 Rationale ................................................................................................................................. 3 1.4 Preliminary data ...................................................................................................................... 4 1.5 Aim and Objectives ................................................................................................................. 5 1.6 Dissertation Structure ............................................................................................................. 5 CHAPTER 2 Literature Review ............................................................................................................. 6 2.1 Introduction ............................................................................................................................ 6 2.2 Molecular Biology ................................................................................................................... 6 2.3 Classification of rotavirus ........................................................................................................ 9 2.4 Replication and Pathogenesis ............................................................................................... 10 2.5 Epidemiology and Prevalence ............................................................................................... 11 2.6 Immune response ................................................................................................................. 13 2.7 Treatment ............................................................................................................................. 14 2.8 Vaccines ................................................................................................................................ 15 vi 2.8.1 Live attenuated rotavirus vaccines ............................................................................... 15 2.8.2 Non-live rotavirus vaccines ........................................................................................... 19 2.9 Heterologous gene expression ............................................................................................. 21 CHAPTER 3 Construction of dual expression vectors containing yeast codon-optimized sequences encoding rotavirus proteins VP2 and VP6........................................................................ 23 3.1 Introduction .......................................................................................................................... 23 3.2 Materials and Methods ......................................................................................................... 24 3.2.1 Enzymes, kits, general chemicals and reagents ............................................................ 24 3.2.2 Strains of bacteria and cultivation medium .................................................................. 24 3.2.3 General methods .......................................................................................................... 25 3.2.4 Vectors and rotavirus strain .......................................................................................... 28 3.2.5 Yeast codon-optimized ORFs ........................................................................................ 29 3.2.6 Cloning strategy ............................................................................................................ 31 3.3 Results and Discussion .......................................................................................................... 35 3.3.1 Cloning Approach .......................................................................................................... 35 3.3.3 Yeast codon-optimized ORFs ........................................................................................ 38 3.3.4 Construction of expression vectors containing yeast optimized rotavirus VP2 and VP6 ORFs …………………………………………………………………………………………………………………………………40 3.3.5 Construction of dual expression vectors containing both VP2 and VP6 ORFs in single vectors…………………………………………………………………………………………………………………………………….43 3.4 Summary ............................................................................................................................... 45 CHAPTER 4 Engineering recombinant yeast strains and expression of rotavirus VP6 protein in yeast . 46 4.1 Introduction .......................................................................................................................... 46 4.2 Materials and Methods ......................................................................................................... 47 4.2.1 Enzymes, kits, general chemicals and reagents ............................................................ 47 4.2.2 Yeasts strains and mammalian cells and cultivation medium ...................................... 47 4.2.3 General methods .......................................................................................................... 49 4.2.4 Engineering of recombinant yeast strains .................................................................... 51 4.2.5 Rotavirus VP6 protein expression ................................................................................
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