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Thesis Martina Decker, Characterization EH3 & EH4 Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2010 Expression and characterization of the novel human epoxide hydrolases EH3 and EH4 Decker, Martina Marion Elisabeth Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-46370 Dissertation Published Version Originally published at: Decker, Martina Marion Elisabeth. Expression and characterization of the novel human epoxide hydro- lases EH3 and EH4. 2010, University of Zurich, Faculty of Science. EXPRESSION AND CHARACTERIZATION OF THE NOVEL HUMAN EPOXIDE HYDROLASES EH3 AND EH4 DISSERTATION zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Martina Marion Elisabeth Decker aus Deutschland Promotionskomitee Prof. Dr. Michael Arand Prof. Dr. Jean-Marc Fritschy Prof. Dr. Thierry Hennet Zürich, 2010 2 3 4 ACKNOWLEDGEMENTS First, I would like to thank Michael Arand for supervising me and giving me the opportunity to do my PhD in his group. Many thanks for sending me to San Diego and Baltimore (the best steak ever) and of course for the intensive proofreading and his help during writing this thesis. I would also like to thank “Miss Annette” Cronin for simply everything. For helping me with any problems in the lab and outside the lab, for answering all my questions on LC-MS/MS and protein purification and for wonderful barbecues with wonderful potato salad. It was great going to Las Vegas and Baltimore with you! I am really going to miss you. Also a big thank you to Magdalena Adamska, for introducing me into the secrets of molecular biology and for granting me asylum in the very beginning of my PhD. Also for being there and backing me up whenever I had the feeling that something was wrong. Many thanks go to all the people of the Toxi group, those who are still there and those who are already gone. In particular to Severine for all the nice and relaxing “coffee and cigarettes” and for going to Kondi, which always helps to keep in shape. Thanks a lot to Kathrin - Katruschka for keeping an eye on insect cells and for constantly providing me with fresh and lively cells! Also thanks a lot to my “Masters of Desasters” - Janine and Francesca. Thank you for the nice time, the help and support in the lab, for cheering me up and for going to Sauna. I would also like to thank my office buddy Marc, for all the delicious mensa meals we had, for making me laugh and for being there even when everyone else already has finished work. Also “merci vielmals” to Jean-Mary Fritschy, who always responded to emails immediately and helped me with all the difficult questions about “Zirkulationskreise”, “Gutachten” and other important paperwork. Thanks a million also to all the people outside the institute, who made it possible for me to accomplish my PhD. Especially to Oli and Wibi, for giving me advise and for always having an ear, for having drinks and fun, for playing handball together with me and for being such friends. I also want to thank my parents who always cared and always supported me. Thanks for welcoming me at home whenever I was homesick and felt like going back to the roots. Thanks for patiently coping with my bad moods. Thanks for always believing in me and for being proud of me. Last but not least I would like to thank two very important people in my life, Katharina and Philipp. I would probably have never been able to finish this PhD without them. Thank you very much for believing in me, supporting me, loving me, listen to me, being there for me whenever I desperately needed someone. I know I could have always called you. This is impayable. 5 6 TABLE OF CONTENTS ACKNOWLEDGEMENTS ....................................................................................................................... 5 TABLE OF CONTENTS .......................................................................................................................... 7 1 SUMMARY / ZUSAMMENFASSUNG ...................................................................................... 11 2 INTRODUCTION ...................................................................................................................... 15 2.1 Epoxide hydrolases ................................................................................................................ 15 2.1.1 The α/β hydrolase fold family.................................................................................................... 16 2.1.2 Mechanism of epoxide hydrolysis ............................................................................................. 18 2.2 Function and human relevance of sEH and mEH ............................................................... 19 2.2.1 Epoxide hydrolysis - role in detoxification ................................................................................. 20 2.2.2 Epoxide hydrolysis - role in physiology ..................................................................................... 21 2.3 sEH as pharmacological target - Development of sEHi ...................................................... 23 2.4 Identification of novel epoxide hydrolases .......................................................................... 24 3 AIM OF THE STUDY ................................................................................................................ 27 4 MATERIALS AND METHODS ................................................................................................. 29 4.1 Chemicals and enzymes ........................................................................................................ 29 4.2 Vectors and plasmids ............................................................................................................. 29 4.3 Basic molecular biology methods ........................................................................................ 29 4.3.1 PCR .......................................................................................................................................... 29 4.3.2 TOPO cloning ........................................................................................................................... 30 4.3.3 Transformation and plasmid amplification ................................................................................ 30 4.3.4 Plasmid isolation ....................................................................................................................... 30 4.3.5 Analyzing plasmid ..................................................................................................................... 30 4.3.6 Cloning of cDNA into target vector ........................................................................................... 31 4.4 mRNA isolation ....................................................................................................................... 31 4.5 cDNA synthesis ...................................................................................................................... 31 4.6 Recombinant protein expression .......................................................................................... 31 4.6.1 Recombinant expression in E. coli ........................................................................................... 31 4.6.2 Recombinant expression in mammalian cells .......................................................................... 32 4.6.2.1 Cultivation of mammalian cells ............................................................................................ 32 4.6.2.2 Stable transfection ............................................................................................................... 32 4.6.3 Recombinant expression in insect cells .................................................................................... 32 4.6.3.1 Generation of Bacmid .......................................................................................................... 33 4.6.3.2 Transfection of insect (Sf9) cells ......................................................................................... 34 4.6.3.3 Routine cultivation and expression of recombinant protein ................................................. 34 4.7 Protein analysis and characterization .................................................................................. 34 4.7.1 SDS-PAGE ............................................................................................................................... 34 4.7.2 Coomassie staining .................................................................................................................. 35 4.7.3 Western blot analysis ................................................................................................................ 36 4.7.4 Protein purification .................................................................................................................... 36 4.7.4.1 GST affinity chromatography ............................................................................................... 36 4.7.4.2 Affinity chromatography of His-tagged proteins .................................................................. 36 4.7.5 Peptide Analysis by LC-MS/MS ................................................................................................ 37 4.7.5.1 Sample preparation from Sf9 culture (methanol extraction) ................................................ 37 4.7.5.2 Sample
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