Tobacco shoot regeneration from calli in temporary immersion culture for biosynthesis of heterologous biopharmaceuticals Sherwin Savio Barretto Thesis submitted for the Degree of Doctor of Philosophy PhD Imperial College London Department of Life Sciences Faculty of Natural Sciences Imperial College London 2014 Declaration of Originality I hereby declare that this thesis, submitted in fulfilment of the requirements for the degree of Doctor of Philosophy of Imperial College London, represents my own work and has not been previously submitted to this or any other institute for any degree, diploma or other qualification. Sherwin Savio Barretto 1 Copyright Declaration The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work. 2 Abstract ‘Molecular farming’, the use of transgenic plants to produce biopharmaceutical proteins is emerging as a new biotechnological paradigm. Transgenic plants offer several advantages over conventional microbial and mammalian cell host technologies. In particular, transplastomic plants, with transformed plastid genomes, are capable of massive expression of foreign proteins and represent a promising platform for biopharmaceutical synthesis. The main theme of this PhD thesis is the investigation of in vitro regeneration of tobacco (Nicotiana tabacum) shoots from callus tissue in temporary immersion (TI) culture for heterologous biopharmaceutical synthesis. There is special emphasis on subunit vaccine expression in transplastomic tobacco, in which foreign protein accumulation is correlated with chloroplast number and development during the organogenesis process. Studies using transplastomic N. tabacum expressing TetC (tetanus toxin fragment C) investigated the influence of several culture parameters on biomass regeneration and recombinant protein expression. The parameters investigated include medium nitrogen source ratio, sucrose concentration and hydrodynamics. These studies highlight the sensitivity of transplastomic protein yields to the culture microenvironment, and provide a starting point for further optimisation. Further studies demonstrated the feasibility of TI culture for biosynthesis of proteolytically-unstable transplastomic subunit vaccines, p24 (HIV antigen) and VP6 (rotavirus antigen). TI culture is also demonstrated as a means for nuclear expression of functional Guy’s 13 monoclonal antibody. Finally, the use of TI culture as the basis of novel technological innovations is investigated. This includes the demonstration of transplastomic protein expression in a prototype large-scale mechanical temporary immersion bioreactor. Encapsulation of callus aggregates in an alginate matrix for long-term germplasm preservation was trialled, prior to temporary immersion regeneration. Overall, this work presents a novel in vitro propagation method for the contained large-scale biosynthesis of biopharmaceutical proteins, as a potential alternative to conventional plant propagation platforms based on agricultural cultivation or cell suspension culture. 3 Acknowledgements I gratefully acknowledge all the individuals who have provided the assistance and support I needed to complete this complex assignment. First of all, I would like to express my sincere gratitude and deep appreciation to my supervisor, Prof. Peter Nixon, for providing me with the opportunity to pursue this exciting and challenging PhD project, and for all his guidance and support. I am equally grateful to my co-supervisor Prof. Klaus Hellgardt whose guidance and invaluable technical ‘know- how’ has helped me tremendously. I would like to give my deepest thanks to Dr Franck Michoux, the post-doc who started this project and who in many ways has acted as my ‘unofficial supervisor’. I am extremely grateful for his assistance, support, time and patience. I would like to thank all members of the 7th floor of the Ernst Chain Building, past and present, for their cooperation, encouragement, support and friendship. I would especially like to thank Hussain Haji Taha, Dr Jianfeng Yu, Shengxi Shao, Dr Niaz Ahmad, Dr Steven Burgess, Dr Marko Boehm, Dr Charlotte Ward, Dr Agripina Banda, Sana Asghar, Xu Zhao, Alexandros Papagiannakis, Jiyao Gan, Chi Zeng, Zheng-Yi Wei, Jayasudha Nagarajan, Marin Sawa, Charlie Cotton, Jeffery Douglass, Sven Dc, Dr Tanai Cardona Londono, Dr Karim Maghlaoui, Dr Alison Telfer, Katharina Brinkert, Dr Wojciech Bialek, Dr Andreas Fantuzzi, Dr Gillian Young, Ruiqiong An, Dr Masooma Rasheed, Ewelina Krysztofinska, Mostafa Jamshidiha, Dr Lisa Hale, Dr Justin Yeoman, Dr James MacDonald, Bhavish Patel, Dr Christian Richard, Amanda Koslovaite and all other lab-fellows, past and present, whom I have had the greatest pleasure crossing paths with. In particular, I will always remember Prof. Bill Rutherford for his carefree attitude, advice and chats. I would like to thank all my collaborators, for their help, guidance, materials and equipment. I would like to thank Prof. John Gray, University of Cambridge for providing me with the transplastomic seeds for the expression of p24 and VP6 and the accompanying antibodies. I would like to thank Prof. Julian Ma and Pascal Drake for providing me with the transformant seeds for the expression of Guy’s 13 monoclonal antibody. These collaborative efforts have helped in the advancement of the scientific endeavour. 4 I would like to thank the Biotechnology and Biological Sciences Research Council (BBSRC) Targeted Priority Studentships initiative for funding this work. I am deeply indebted to my family for their love, support, encouragement and prayers for my success. Finally, I would like to give thanks to Almighty God, for the innumerable graces and blessings bestowed upon me to enable me to undertake this pursuit. 5 Table of Contents Table of Contents .................................................................................................................................... 6 List of Figures ........................................................................................................................................ 14 List of Tables ......................................................................................................................................... 17 List of Abbreviations ............................................................................................................................. 18 Chapter 1. Introduction .................................................................................................................. 23 1.1 The Biotechnology revolution and recombinant biopharmaceuticals ................................. 23 1.2 Transgenic plants .................................................................................................................. 25 1.2.1 The scope of transgenic plants as an alternative host technology ............................... 25 1.2.2 The benefits of transgenic plant host systems relative to conventional platforms ..... 27 1.2.3 Bioprocessing of plant-derived biopharmaceuticals .................................................... 29 1.2.3.1 Choice of localisation target in transgenic plant host systems ................................. 29 1.2.3.2 The choice of in vitro or soil-based cultivation for growth of transgenic plants ...... 30 1.2.3.3 The use of bioreactors in cell and tissue cultures ..................................................... 32 1.2.3.4 The temporary immersion culture format ................................................................ 33 1.2.3.5 Downstream processing ........................................................................................... 37 1.2.4 Chloroplast transformation ........................................................................................... 38 1.2.4.1 An overview of plant genetic transformation strategies .......................................... 38 1.2.4.2 The plastid genome as a target for genetic transformation ..................................... 39 1.2.4.2.1 Plastids organelles in higher plants .................................................................... 39 1.2.4.2.2 Features of the plastid genome .......................................................................... 40 1.2.4.2.3 The plastid genome as a novel target for genetic engineering .......................... 42 1.2.4.2.4 Methods for transformation of the chloroplast ................................................ 42 1.2.4.2.5 Benefits of plastid transformation ...................................................................... 44 1.2.4.2.5.1 Hyperexpression of transgenic proteins in plastids ..................................... 44 6 1.2.4.2.5.2 Post-transcriptional regulation of plastid protein synthesis and implications for transformation vectors ................................................................................................ 45 1.2.4.2.5.3 Polycistronic transcription of plastid genes and opportunities for metabolic pathway engineering ........................................................................................................ 46 1.2.4.2.5.4 Maternal inheritance of the chloroplast genome and implications for biosafety 47 1.2.4.2.6