Detection of novel serological biomarkers in two neurodegenerative disorders: Morbus Alzheimer and Multiple Sclerosis Inaugural-Dissertation To obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) Submitted to the Department of Biology, Chemistry and Pharmacy of Freie Universität Berlin by Yuliya Georgieva born in Orenburg, Russian Federation Oktober 2013 I hereby declare that all work and writing contained within this thesis was conducted by myself, all used references are cited accordingly and any personal assistance has been acknowledged by name. All work for this thesis was conducted from June 2009 to June 2013 in the working group of Dr. Zoltán Konthur at the Department of Vertebrate Genomics under the supervision of Prof. Dr. Hans Lehrach in the Max Planck Institute for Molecular Genetics. 1. Reviewer: Prof. Dr. Hans Lehrach Max Planck Institute for Molecular Genetics Department of Vertebrate Genomics Ihnestraße 63-73 14195 Berlin, Germany 2. Reviewer: Prof. Dr. Burghardt Wittig Freie Universität Berlin Foundation Institute Molecular Biology and Bioinformatics Arnimallee 22 14195 Berlin, Germany Date of defense: 14.02.2014. Acknowledgment I would like to thank Prof. Dr. Hans Lehrach, Director at the Max Planck Institute for Molecular Genetics, for giving me the great opportunity to conduct my dissertation at his department in such an interesting research area. Furthermore, I thank Prof. Dr. Burghardt Wittig from Freie Universität Berlin for his kindness to serve as an academic advisor of my thesis and for his invaluable advice on this manuscript. I would like to express my sincere gratitude to my direct supervisor Dr. Zoltán Konthur for the support of my dissertation, for his scientific advice, as well as for giving me the opportunity to take part at international conferences and for enabling my voluntary work at btS e.V.. I also would like to thank all the members of AG Konthur, current and previous, for their helpful hands and many inspiring discussions. In particular, my two companion Ph.D. students Dr. Stephan Klatt and Dr. Florian Rubelt, as well as Carola Stoschek, for great working atmosphere, support and amity. I especially want to thank my student assistant Sabrina Grasse for her precious assistance during most of the experimental work and her close friendship. My sincere thanks go to our project cooperation partner, Dr. Steffen Hennig for his collaboration and advice in the field of bioinformatics and next generation sequencing. I would also like to thank Dr. Matthias Kaup for the critical revision of this manuscript and for his scientific advice. Last but not least, I would like to thank my parents Svetlana and Ivan Georgievi, my brothers Georgi and Dimo Georgievi and all my dear friends for their invaluable support, patience and encouraging in dire straits. Table of Contents Acknowledgement ...................................................................................................................... 3 List of abbreviations ................................................................................................................... 9 1. Introduction........................................................................................................................... 14 1.1. Diagnostic aspects in neurodegenerative disorders ...................................................... 16 1.1.1. Morbus Alzheimer (AD) diagnostics ................................................................... 16 1.1.2. Multiple sclerosis (MS) diagnostics..................................................................... 22 1.1.3. Serological autoimmunity-based biomarkers...................................................... 25 1.2. High-throughput screening technologies ....................................................................... 28 1.2.1. Protein macroarray technology........................................................................... 29 1.2.2. Filamentous phage display of full-ORF polypeptides ......................................... 31 1.2.3. Next generation sequencing ............................................................................... 36 2. Objective .............................................................................................................................. 40 3. Materials............................................................................................................................... 42 3.1. Consumables................................................................................................................. 42 3.2. Chemicals, buffers and solutions................................................................................... 43 3.3. Growth culture media and additives............................................................................... 46 3.4. E. coli strains.................................................................................................................. 47 3.5. Helper phages................................................................................................................ 47 3.6. Plasmids and OC source libraries.................................................................................. 47 3.7. Protein macroarrays....................................................................................................... 48 3.8. Human blood sera.......................................................................................................... 48 3.9. Antibodies ...................................................................................................................... 50 3.10. Magnetic beads and affinity columns........................................................................... 51 3.11. DNA and Protein markers............................................................................................ 51 3.12. Kits............................................................................................................................... 52 3.13. Enzymes ...................................................................................................................... 52 3.14. Oligonucleotides .......................................................................................................... 53 4 3.15. Laboratory equipment.................................................................................................. 54 3.16. Software....................................................................................................................... 55 4. Methods................................................................................................................................ 57 4.1. Molecular biology based methods ................................................................................. 57 4.1.1. DNA purification.................................................................................................. 57 4.1.1.1. DNA extraction from agarose gel ......................................................... 57 4.1.1.2. DNA precipitation with ethanol ............................................................. 57 4.1.1.3. Plasmid purification .............................................................................. 57 4.1.2. DNA digestion with restriction enzymes ............................................................. 58 4.1.3. Dephosphorylation of digested plasmids ............................................................ 58 4.1.4. DNA separation in agarose gel electrophoresis ................................................. 58 4.1.5. DNA amplification by polymerase chain reaction (PCR) .................................... 58 4.1.6. DNA ligation........................................................................................................ 59 4.1.7. Gateway LR recombination ............................................................................... 60 4.1.8. DNA Sanger sequencing .................................................................................... 61 4.1.9. Preparation of electro-competent E. coli cells .................................................... 61 4.1.10. Transformation of E. coli cells by electroporation ............................................. 61 4.2. Protein biochemistry based methods............................................................................. 62 4.2.1. IPTG-induced protein expression in E.coli cells ................................................. 62 4.2.2. Protein extraction under native and denatured conditions.................................. 62 4.2.2.1. Cytoplasmic protein extraction ............................................................. 62 4.2.2.2. Periplasmic protein extraction .............................................................. 63 4.2.3. IMAC purification of recombinant His6-tagged proteins ...................................... 63 4.2.3.1. Purification with Ni-NTA-Agarose at gravity flow (batch purification) ... 63 4.2.3.2. Purification with Ni-NTA columns on an FPLC system ........................ 63 4.2.4. Concentrating proteins........................................................................................ 64 4.2.5. Protein separation in SDS PAGE ....................................................................... 64 4.2.6. Coomassie staining ............................................................................................ 64 4.2.7. Silver staining ..................................................................................................... 64 5 4.2.8. Western Blot ......................................................................................................