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Avidin As an Alternative Scaffold Acta Universitatis Tamperensis 2305 SOILI LEHTONEN Avidin as an Alternative Scaffold Alternative Scaffold as an Avidin SOILI LEHTONEN Acta Universitatis Tamperensis 2305 SOILI LEHTONEN Avidin as an Alternative Scaffold Development of avidin-based small molecule binding proteins, antidins AUT 2305 AUT SOILI LEHTONEN Avidin as an Alternative Scaffold Development of avidin-based small molecule binding proteins, antidins ACADEMIC DISSERTATION To be presented, with the permission of the Faculty Council of the Faculty of Medicine and Life Sciences of the University of Tampere, for public discussion in the auditorium F115 of the Arvo building, Arvo Ylpön katu 34, Tampere, on 15 September 2017, at 12 o’clock. UNIVERSITY OF TAMPERE SOILI LEHTONEN Avidin as an Alternative Scaffold Development of avidin-based small molecule binding proteins, antidins Acta Universitatis Tamperensis 2305 Tampere University Press Tampere 2017 ACADEMIC DISSERTATION University of Tampere, Faculty of Medicine and Life Sciences National Doctoral Programme in Informational and Structural Biology (ISB) Doctoral Programme in Biomedicine and Biotechnology Finland Supervised by Reviewed by Professor Emeritus Markku Kulomaa Docent Urpo Lamminmäki University of Tampere Tampere University of Technology Finland Finland Associate Professor Vesa Hytönen Professor Stefan Ståhl University of Tampere KTH Biotechnology Royal Institute of Technology Finland Sweden The originality of this thesis has been checked using the Turnitin OriginalityCheck service in accordance with the quality management system of the University of Tampere. Copyright ©2017 Tampere University Press and the author Cover design by Mikko Reinikka Acta Universitatis Tamperensis 2305 Acta Electronica Universitatis Tamperensis 1809 ISBN 978-952-03-0513-0 (print) ISBN 978-952-03-0514-7 (pdf) ISSN-L 1455-1616 ISSN 1456-954X ISSN 1455-1616 http://tampub.uta.fi Suomen Yliopistopaino Oy – Juvenes Print Tampere 2017 441 729 Painotuote This PhD thesis is dedicated to our dear children: Juho, you are the sunshine of my life. Don’t lose the enthusiasm you have for everything! And our dear unborn baby, take your time to develop despite the defense. We’ll meet you soon! ACKNOWLEDGEMENTS My deepest gratitude goes to my supervisors Professor Emeritus Markku “Kuku” Kulomaa and Associate Professor Vesa Hytönen. Kuku welcomed me to his group and has provided me many interesting opportunities to broaden my knowledge and grow as a scientist through the years. Thank you, Kuku, for your professional support and for providing a warm, pleasant working atmosphere. I wish to thank my other supervisor Vesa for professional guidance and everlasting optimism. Your enthusiastic attitude towards science and your endless thirst for knowledge has been very inspiring. Additionally, I want to thank you both for the first-class working environment and facilities. I am thankful for Dr. Tiina Riihimäki for introducing me to the fascinating world of phage display and kindling my interest in protein engineering. Thank you for encouraging and teaching me. I also want to thank Dr. Barbara Taskinen for brainstorming and successful collaboration on the DNA library design and phage display. You two have been excellent coworkers, and without you this work would not have been possible! I would also like to thank Antti Tullila and Dr. Tarja Nevanen from VTT Technical Research Center of Finland for sharing ideas and successful collaboration. I also want to thank Sampo Kukkurainen for great work with the modelling and simulations, as well as for creating the Perl script for analyzing the DNA sequencing results. Further thanks go to Dr. Purvi Jain who later joined the group for half a year and taught me new tricks in phage display. During these years I have had the pleasure to supervise three Master’s theses and a Bachelor’s thesis, as well as to guide several summer students. Thank you Elina Ojala, Rolle Rahikainen, Niklas Kähkönen, Sandra Posch, and Meri Uusi-Mäkelä, who were eager and talented students and contributed to my different projects. I wish to express my gratitude to Ulla Kiiskinen, Niklas Kähkönen, Outi Väätäinen, Latifeh Azizi, and Laura Kananen for the excellent technical support and endless willingness to help. Special thanks to my dear “lunch buddies” Dr. Tiina Riihimäki, Dr. Juha Määttä, Dr. Tiia Koho, Dr. Jenni Leppiniemi, Rolle Rahikainen, Dr. Jenita Pärssinen, Dr. Sanna Auer, Dr. Minna Hankaniemi, Niila Saarinen, Magdaléna von Essen and Latifeh Azizi for enjoyable conversations (about work and everything else) and good times over these years. It has been a pleasure working with you! I would like to also thank other MBT and PD group members, the new and former ones: the late Docent Henri Nordlund, Dr. Satu Helppolainen, Dr. Jarkko Valjakka, Dr. Olli Laitinen, Dr. Anssi Mähönen, Anssi Nurminen, Dr. Inger Vikholm-Lundin, Dr. Vasyl Mykuliak and various students over the years for creating an excellent working environment. I am grateful to my thesis committee members Professor Emeritus Pentti Tuohimaa, Professor Kristiina Takkinen, and Dr. h.c. Jouko Haapalahti for interesting discussions and valuable comments during my thesis project. Additionally, I wish to express my sincere gratitude to all my collaborators and co- authors not already mentioned: Dr. Martina Rangl, Dr. Andreas Ebner, Professor Peter Hinterdorfer, Masi Koskinen, Nitin Agrawal, Professor Mark Johnsson, and Dr. Tomi Airenne. I would like to thank my reviewers Professor Stefan Ståhl and Dr. Urpo Lamminmäki for their valuable comments and feedback to improve my thesis, and Dr. Eloise Mikkonen for proofreading the English. I also would like to thank the Academy of Finland (grants to M.S.K.) and the National Doctoral Programme in Informational and Structural Biology (ISB) for funding, and a personal grant from the Finnish Cultural Foundation for finalizing the thesis. In addition to funding, ISB also enabled me to make connections with other graduate students throughout Finland by organizing excellent annual Spring and Winter meetings. Furthermore, I would like to thank the Scientific Foundation of the City of Tampere for a grant for printing this PhD thesis. I also thank the Pirkanmaa Hospital District for financial support. The balance in my life has come from my dear friends with whom I have forgotten the stress of work. Thank you all for enjoyable moments during these years: Anu, Jouni, Kati, Vesku, Elli, Jussi, Sami, Kaisa, Hanna, Mikko, Sari, Henna, Laura, Jouni, Jossu, Joonas, Anna, Suvi, and Aino. I am thankful for the numerous good times and experiences we have shared together! Last but not least, I am grateful to my family, relatives, and family-in-law for their support and care over the years. I wish to communicate a great expression of gratitude to my parents for all their support and encouragement during my studies. I also want to thank my parents-in-law for their hospitality and all their help. Help from both parents has been priceless during the last couple of years. Kiitokset tuesta ja muistamisista rakkaille Mummuilleni! Thank you Antti, Johanna, Mikko, and Terhi for your hospitality and enjoyable times. Finally, my deepest gratitude goes to my dear husband Jukka and our dear son Juho. You bring so much love, joy, and happiness to my life. Thank you, Jukka, for sharing your life with me, being my best friend, patiently supporting, encouraging, and loving me. I love you! Tampere, August 2017 ABSTRACT Antibodies, the specific binding proteins produced by our immune system for virtually any foreign target molecule, have been used as valuable laboratory reagents in the life sciences and as therapeutic drugs in medicine for almost a century. However, in recent years these antibody-based affinity reagents have been challenged by novel types of binding proteins developed through directed evolution, offering preferable properties in certain applications. The aim of this thesis was to develop avidin scaffold-based novel binding proteins for small molecule targets, which are challenging to develop high-affinity antibodies for. Avidin is an exceptionally stable protein from chicken egg-white, known for its high affinity towards its native ligand, biotin (Kd~10-15M). This is the srongest non-covalent interaction known to nature between a protein-ligand pair. The structure-function of avidin is well known, and avidin is known to tolerate genetic modifications well, especially when located in the loop regions. Above all, lipocalins belonging to the same “calycin” superfamily as avidin, have already been successfully used as alternative scaffolds. Avidin libraries were constructed by targeting loop region amino acid residues involved in biotin binding for randomization, and phage display was used for selection. Testosterone was chosen as the first target molecule, resulting in the selection of avidin variants, antidins, with micromolar affinities. With the help of affinity maturation, the remaining biotin-binding affinity was signicantly reduced while improving the affinity towards testosterone. Later several avidin libraries were pooled together and used for the selection of binders towards several diagnostically relevant small molecules in parallel. The obtained results showed that an avidin scaffold has potential to be used as an alternative scaffold for several small molecules. As a result of this PhD project, new methods for library construction were established. The new Gateway-compatible phagemid (pGWphagemid) vector was constructed to enable more efficient library generation utilizing the homologous
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