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Struktur-Funktionsbeziehungen Der Rho- Und Der Plexin-Proteinfamilien

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Struktur-Funktionsbeziehungen Der Rho- Und Der Plexin-Proteinfamilien Struktur-Funktionsbeziehungen der Rho- und der Plexin-Proteinfamilien Inauguraldissertation zur Erlangung des Doktorgrades der Fakultät für Chemie der Ruhr-Universität Bochum vorgelegt von Dennis Franz-Josef Fiegen aus Kleve April 2005 INHALTSVERZEICHNIS _______________________________________________________________________________________________________________________________________________________________________________________________________________ Inhaltsverzeichnis Inhaltsverzeichnis .................................................................................................................... III Abbildungsverzeichnis ............................................................................................................ IX Tabellenverzeichnis ................................................................................................................. XI Abkürzungsverzeichnis ..........................................................................................................XII 1. Einleitung ........................................................................................................................ 1 1.1 GTP-bindende Proteine als Elemente der Signaltransduktion ..................................... 2 1.2 Die Ras-Superfamilie ..................................................................................................... 3 1.3 Die GTPasen der Rho-Familie ....................................................................................... 7 1.4 Regulatoren und Effektoren der RhoGTPasen ............................................................... 8 1.5 Die Signaltransduktion der Plexin-Transmembranrezeptoren........................................ 15 2. Zielsetzung ..................................................................................................................... 21 3. Material und Methoden ................................................................................................ 22 3.1 Material ........................................................................................................................... 22 3.1.1 Bakterienstämme ...................................................................................................22 3.1.2 Insektenzelllinien ................................................................................................22 3.1.3 Nährmedien und Antibiotika ................................................................................ 23 3.1.3.1 Nährmedien und Zusätze ......................................................................... 23 3.1.3.2 Antibiotika ..............................................................................................23 3.1.4 Puffer und Lösungen ............................................................................................ 23 3.1.5 Vektoren ..............................................................................................................27 3.1.6 Oligonukleotide ...................................................................................................28 3.1.6.1 Oligonukleotide zur Fragmentherstellung ............................................... 28 3.1.6.2 Oligonukleotide zur Sequenzierung ........................................................ 29 3.1.7 Biochemikalien und Chemikalien ......................................................................... 30 3.1.7.1 Proteine und Enzyme ................................................................................ 30 3.1.7.2 Nukleotide ................................................................................................30 3.1.7.3 Protein- und Nukleinsäurestandards ........................................................ 31 3.1.7.4 Proteinase-Inhibitoren .............................................................................31 3.1.7.5 FPLC-Säulenmaterial .............................................................................31 3.1.7.6 Chemikalien ..............................................................................................31 3.1.7.7 Antikörper ................................................................................................32 _______________________________________________________________________________________ III Struktur-Funktionsbeziehungen der Rho- und der Plexin-Proteinfamilien INHALTSVERZEICHNIS _______________________________________________________________________________________________________________________________________________________________________________________________________________ 3.1.8 Kit-Systeme .......................................................................................................... 32 3.1.9 Geräte ................................................................................................................... 32 3.1.10 Verbrauchsmaterialien ....................................................................................... 33 3.1.11 Kristallographische Screens ................................................................................ 34 3.1.12 Programme .......................................................................................................... 34 3.2 Molekularbiologische Methoden .................................................................................. 35 3.2.1 Herstellung kompetenter E. coli Zellen ............................................................... 35 3.2.2 Transformation von E. coli .................................................................................. 35 3.2.3 Kultivierung und Lagerung von transformierten Bakterienzellen ....................... 35 3.2.4 Isolierung von Plasmid-DNA ................................................................................ 36 3.2.5 Präparation von Bakmid-DNA ............................................................................. 36 3.2.6 Hydrolyse von Plasmid-DNA mit Restriktionsendonukleasen ............................ 37 3.2.7 Agarose-Gelelektrophorese .................................................................................. 37 3.2.8 Isolierung von DNA-Fragmenten aus Agarose-Gelen .......................................... 38 3.2.9 Ligation ................................................................................................................. 38 3.2.10 Polymerasekettenreaktion (PCR) ...................................................................... 38 3.2.11 Analytische Schnell-PCR zur Bestimmung positiver Klone .............................. 40 3.2.12 DNA-Sequenzierung ......................................................................................... 41 3.3 Insektenzellen ................................................................................................................. 42 3.3.1 Kulturbedingungen .............................................................................................. 42 3.3.2 Transfektion von Sf21-Zellen mit rekombinanter Bakmid-DNA ....................... 42 3.3.3 Bestimmung des Virus-Titers ............................................................................. 43 3.3.4 Virusamplifikation ................................................................................................ 44 3.3.5 Proteinexpression in Sf21-Zellen ......................................................................... 44 3.4 Proteinchemische Methoden ......................................................................................... 45 3.4.1 Analytische Expression rekombinanter Plasmide ................................................. 45 3.4.2 Analytischer Aufschluss von Bakterienzellen ...................................................... 45 3.4.3 Präparative Expression rekombinanter Proteine.................................................... 45 3.4.4 Präparativer Aufschluss von Bakterienzellen ...................................................... 46 3.4.5 Präparativer und analytischer Aufschluss von Insektenzellen .............................. 46 3.4.6 Affinitätschromatographie .................................................................................. 46 3.4.6.1 GST Gene Fusion System ......................................................................... 47 3.4.6.2 QIAexpress System .................................................................................. 47 3.4.7 Ionenaustauscherchromatographie ...................................................................... 47 _______________________________________________________________________________________ Struktur-Funktionsbeziehungen der Rho- und der Plexin-Proteinfamilien IV INHALTSVERZEICHNIS _______________________________________________________________________________________________________________________________________________________________________________________________________________ 3.4.8 Gelfiltration .......................................................................................................... 48 3.4.9 Protease-Verdau von GST-/6xHis-Fusionsproteinen .......................................... 48 3.4.10 Ultrafiltration zur Konzentrierung ...................................................................... 49 3.4.11 Konzentrationsbestimmung nach Bradford ........................................................ 49 3.4.12 SDS-Polyacrylamid-Gelelektrophorese (SDS-PAGE)........................................ 49 3.4.13 Western Blot ....................................................................................................... 51 3.4.14 Umkehrphasen HPLC ........................................................................................
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