Generation of novel intracellular binding reagents based on the human γB-crystallin scaffold Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) vorgelegt der Naturwissenschaftlichen Fakultät I-Biowissenschaften der Martin-Luther-Universität Halle-Wittenberg Institut für Biochemie und Biotechnologie von Ewa Mirecka geboren am 17. Dezember 1976 in Gdynia, Polen Table of contents Table of contents 1. INTRODUCTION.........................................................................................................1 1.1 Monoclonal antibodies as a biomolecular scaffold..........................................................1 1.2 Binding molecules derived from non-immunoglobulin scaffolds.....................................3 1.2.1 Alternative protein scaffolds – general considerations............................................................ 3 1.2.2 Application of alternative binding molecules ........................................................................... 6 1.3 Affilin – novel binding molecules based on the human γB-crystallin scaffold................... 6 1.3.1 Human γB-crystallin as a molecular scaffold........................................................................... 6 1.3.2 Generation of a human γB-crystallin library and selection of first-generation Affilin molecules ................................................................................................................................ 8 1.4 Selection of binding proteins by phage display .................................................................... 9 1.5 Human papillomavirus E7 protein as a model target for the generation of intracellular Affilin molecules...................................................................................................................... 11 1.5.1 Human papillomaviruses and associated diseases .............................................................. 12 1.5.2 Genomic organization of human papillomaviruses ............................................................... 12 1.5.3 Viral life cycle......................................................................................................................... 13 1.5.4 Human papillomavirus E7 protein ......................................................................................... 14 1.5.4.1 Biological activities and cellular targets of the E7 protein ............................................. 14 1.5.4.2 Biochemical characterization of the E7 protein ............................................................. 16 1.6 Options for delivery of therapeutic proteins into mammalian cells................................... 18 1.7 Aim of the study ...................................................................................................................... 20 2. MATERIALS AND METHODS ........................................................................ 21 2.1 Materials ................................................................................................................................... 21 2.1.1 Bacterial strains..................................................................................................................... 21 2.1.2 Eukaryotic cells...................................................................................................................... 21 2.1.3 Phage .................................................................................................................................... 21 2.1.4 Vectors .................................................................................................................................. 22 2.1.5 Oligonucleotides.................................................................................................................... 23 2.1.6 Chemicals.............................................................................................................................. 23 2.1.7 Enzymes................................................................................................................................ 24 2.1.8 Proteins ................................................................................................................................. 24 2.1.9 Antibodies.............................................................................................................................. 24 2.1.10 Kits and standards............................................................................................................. 25 2.1.11 Buffers and solutions......................................................................................................... 25 2.1.12 Media and antibiotics for cultivation of E. coli ................................................................... 26 2.1.13 Media for mammalian cell culture...................................................................................... 27 2.1.14 Chromatography columns ................................................................................................. 27 2.1.15 Laboratory equipment ....................................................................................................... 27 2.1.16 Miscellaneous.................................................................................................................... 28 2.2 Methods.................................................................................................................................... 28 2.2.1 Molecular biology................................................................................................................... 28 2.2.1.1 Plasmid DNA preparation.............................................................................................. 28 2.2.1.2 Determination of DNA concentration............................................................................. 28 2.2.1.3 Polymerase chain reaction ............................................................................................ 28 2.2.1.4 DNA digestion................................................................................................................ 29 2.2.1.5 Generation of double-stranded oligonucleotides........................................................... 29 2.2.1.6 Dephosphorylation of DNA............................................................................................ 30 2.2.1.7 Agarose gel electrophoresis.......................................................................................... 30 2.2.1.8 DNA gel extraction......................................................................................................... 30 2.2.1.9 DNA ligation................................................................................................................... 30 2.2.1.10 Site-directed mutagenesis......................................................................................... 30 2.2.1.11 DNA sequencing........................................................................................................ 31 Table of contents 2.2.2 Transformation of E. coli cells ............................................................................................... 31 2.2.2.1 Chemically competent E. coli cells ................................................................................ 31 2.2.2.2 Electrocompetent E. coli cells ....................................................................................... 31 2.2.3 Phage display selection of anti-E7 Affilin molecules............................................................. 32 2.2.4 Protein expression using recombinant E. coli ....................................................................... 35 2.2.5 Protein purification and chromatographic analysis................................................................ 36 2.2.6 Protein characterization......................................................................................................... 37 2.2.6.1 Quantification of protein concentration.......................................................................... 37 2.2.6.2 SDS-PAGE .................................................................................................................... 38 2.2.6.3 Western blotting............................................................................................................. 38 2.2.6.4 ELISA............................................................................................................................. 38 2.2.6.5 Surface plasmon resonance.......................................................................................... 39 2.2.7 Cell biology ............................................................................................................................ 40 2.2.7.1 Mammalian cell propagation ......................................................................................... 40 2.2.7.2 Tat-mediated internalization of Affilin molecules into mammalian cells ........................ 40 2.2.7.3 Transfection of mammalian cells................................................................................... 41 2.2.7.4 Immunofluorescence microscopy.................................................................................. 41 2.2.7.5 Bromodeoxyuridine incorporation assay ....................................................................... 42 2.2.7.6 Analysis of apoptosis..................................................................................................... 42 3. RESULTS .............................................................................................................................