Evaluation of the protozoan parasite L. tarentolae as a eukaryotic expression host in biomedical research 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 Stephan Klatt born in Braunschweig, Germany April 2013 I hereby declare that all experiments and writing contained within this thesis were conducted by myself, all references used are cited accordingly and any personal assistance has been acknowledged by name. All experiments for this thesis were conducted from October 2008 to April 2013 in the group of Dr. Zoltán Konthur at the Department of Vertebrate Genomics under the supervision of Prof. Dr. Hans Lehrach at 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 Molecular Biology and Bioinformatics Arnimallee 22 14195 Berlin, Germany Date of defence: August 7th 2013 II Acknowledgement 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 in his department. Furthermore, I thank Prof. Dr. Burghardt Wittig from Freie Universität Berlin for his kindness to serve as an academic advisor of my thesis. First and foremost, I would like to express my sincere gratitude to my direct supervisor Dr. Zoltán Konthur for his continuous support, great tutoring, advice and fruitful discussions as well as for the opportunity to travel, meet and collaborate with international scientists. Additionally, I would like to thank Dr. Sylvia Krobitsch for scientific conversations and all the members of the AG Konthur, current and previous, for their helpful hands and scientific exchange. In particular, Carola Stoschek for all her patience and support, Dr. Florian Rubelt, Yuliya Georgieva and Dejan Gagoski for the good working atmosphere, support and friendship. My sincere thanks are also owed to our collaboration partners, Dr. Daniela Hartl and Dr. Daniel Kolarich. Furthermore, I thank Dr. Reinhard Breitling for valuable advice. Moreover, I would like to thank my parents Rotraud and Friedhelm and my friends (for their support and patience). In particular, I want to thank Sina Nowak for proofreading parts of my thesis. Last but not the least I would like to express my great gratitude to Carola, for her deep understanding, support and love. III Table of contents Acknowledgement ..................................................................................................................... III Table of contents ....................................................................................................................... IV List of abbreviations .................................................................................................................. VI 1 Introduction ............................................................................................................................. 9 1.1 General information about Leishmania species ................................................................ 11 1.1.1 The genus Leishmania ......................................................................................... 11 1.1.2 Life cycle and geographical distribution of Leishmania ......................................... 12 1.1.3 Reproduction: Clonality versus sexuality .............................................................. 15 1.1.4 Infectivity and disease pattern of leishmaniasis .................................................... 15 1.1.5 Immune defence and evolutionary acquired mimicry ............................................ 16 1.1.6 Treatment and potential drug targets of Leishmania parasites ............................. 16 1.2 Leishmania tarentolae as biomedical expression host ..................................................... 17 1.2.1 Similarities and differences to human-pathogenic species ................................... 18 1.2.2 Advantages of the protozoan protein expression system ...................................... 19 1.2.3 Cell Culture Conditions and the amastigote-like form ........................................... 19 1.2.4 Posttranslational protein modifications ................................................................. 20 1.2.5 Gene expression and expression vector variants ................................................. 22 2 Aim ........................................................................................................................................ 25 3 Manuscripts ........................................................................................................................... 26 3.1 Manuscript I ..................................................................................................................... 28 3.2 Manuscript II .................................................................................................................... 39 3.2 Manuscript III ................................................................................................................... 54 3.3 Manuscript IV ................................................................................................................... 63 4 Discussion ............................................................................................................................. 73 5 Current state of research and outlook .................................................................................... 81 IV 6 Summary ............................................................................................................................... 83 7 Zusammenfassung ................................................................................................................ 85 8 References ............................................................................................................................ 88 V List of abbreviations 2-D PAGE Two-dimensional poly-acrylamide gel electrophoresis AA Amino-acid AD Alzheimer’s disease APP Amyloid precursor protein Aprt Adenine-phosphorybosyl transferase gene BirA Biotin ligase bp Base pair CAT Chloramphenicol acetyl transferase CHO Chinese Hamster Ovary CL Cutaneous Leishmaniasis CMP Cytidine monophosphate CO2 Carbon dioxide CPB Cysteine protease B CRP C-reactive protein DNA Deoxyribonucleic acid E. coli Escherichia coli eGFP Enhanced green fluorescent protein EPO Erythropoietin FDFT1 Farnesyl-diphosphate farnesyltransferase GPI Glycosylphosphatidylinositol His6-tag Hexahistidine-tag Hsp Heat shock protein VI IFN-γ Interferon-gamma Ig Immunoglobulin JBS Jena Bioscience L. tarentolae Leishmania tarentolae LEXSY BHI Leishmania Expression System, Brain-Heart-infusion based medium LmjF Leishmania major strain Friedlin LPG Lipophosphoglycan LtaP Leishmania tarentolae strain Parrot-TarII ML Mucocutaneous Leishmaniasis MLEE Multilocus enzyme electrophoresis mRNA Messenger ribonucleic acid MS Mass spectrometry mTKIN Cysteine-free mutant of kinesin N Asparagine NW New World Odc Ornithin decarboxylase locus OW Old World P Proline p53 Cellular tumour protein p53 PC4 Serine protease proprotein convertase 4 PNGase F Peptide -N-Glycosidase F PSA Promastigote surface antigen protein PTM Posttranslational protein modification VII Rab GTPase Ras related protein guanosine triphosphate enzyme RNA Ribonucleic acid rRNA Ribosomal ribonucleic acid S Serine S1 Security level 1 S2 Security level 2 sAP1 Secreted acid phosphatase 1 gene sAPPalpha Soluble amyloid precursor protein alpha scFv Single chain fragment variable SITS Species-independent translational sequences SOD1 Cu/Zn Superoxide dismutase SORLA Sorting protein-related receptor containing LDLR class A repeats SP Secretory signal peptide Ssu 18S ribosomal RNA locus T Threonine TEM Transmission electron microscopy TET repressor Tetracycline-controlled transcriptional activation repressor tPA Tissue plasminogen activator TS Trans-Sialidase UTR Untranslated region VL Visceral Leishmaniasis WHO World Health Organization VIII 1 Introduction 1 Introduction The successful use of recombinant proteins as therapeutic agents in human medicine revolutionized biotechnological research in the 1980s [1]. Today, more than 25 years later, recombinant proteins are frequently used, for example, as a treatment of cancer, anaemia and infertility [2], or they form the basis of many diagnostics. The human genome consists of around 23.000 protein-coding genes [3, 4]. The number of different proteins, however, is significantly higher, since alternative mRNA splicing events and posttranslational protein modifications (PTMs) have led to many different proteins emerging from one single gene. For instance, proteins form structural cell elements, transport diverse macromolecules, regulate cellular homeostasis, tag external antigens for primary immune response or are secreted into the extracellular environment. The recombinant therapeutic proteins which are most frequently approved are monoclonal antibodies, proteins with enzymatic activity or generally glycosylated proteins [5-7]. For their production, genetically engineered bacterial organisms, fungi, or mammalian cells are used, which are generally able to correctly fold the protein and synthetize it into a biologically active form [2]. Common pitfalls of many heterologous expression systems are the