Tabakmosaikvirus-Hüllproteinvarianten als Bausteine für die Nano- und Arraytechnologie Von der Fakultät Energie-, Verfahrens- und Biotechnik der Universität Stuttgart zur Erlangung der Würde eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigte Abhandlung vorgelegt von Fania Constanze Geiger aus Heilbronn TECHNISCHE INFORMATIONSBIBLIOTHEK UNIVERSITÄTSBIBLIOTHEK HANNOVER Hauptberichter: PD Dr. Christina Wege Mitberichter: Prof. Dr. Joachim P. Spatz Tag der mündlichen Prüfung: 15. Februar 2010 Biologisches Institut der Universität Stuttgart 2010
Inhaltsverzeichnis Inhaltsverzeichnis 3 Abkürzungsverzeichnis 7 Zusammenfassung 9 Abstract 11 Einleitung 13 Ergebnisse und Diskussion 21 Publikationsmanuskripte in englischer Sprache 30 Tobacco mosaic virus mutants for the assembly of nanorods with programmed longitudinal domains of differently addressable coat proteins 31 Abstract 31 Introduction 32 Material and Methods 35 Construction of TMV-Cys and TMV-Lys particles 35 Virus propagation and Isolation 36 Infectivity and Symptoms of TMV CP mutants 37 Electron microscopy 3 7 RT-PCR ofisolated viral RNA 37 Fluorescent labeling of TMV particles 38 SDS PAGE for the analysis of fluorescently labeled TMV particles 38 CP preparation 38 Assembly of TMV-like nanorods consistlng ofdifferent CP species 39 Electrophoresis of unmodified or labeled TMV-like nanorods 39 Results 40 Construction of p843tmv-cys and p843tmv-lys plasmids 40 Infectivity and Symptoms 41 Particle isolation 41 Verification ofthe mutations on the genomic level 43 Fluorescent labeling of TMV-Lys or TMV-Cys particles 44 3
Assembly of TMV rods with acljacent longitudinal domains of different CP species 45 Performance of unmodified or labeled in vitro assembled particles with distinct protein domains in an electrica! field 46 Discussion 50 Acknowledgements 53 References 54 Coenzyme A-modified tobacco mosaic virus for site-specific protein coupling with the phosphopantetheinyl transferase Sfp from Bacillus subtilis 59 Abstract 59 Introduction 60 Material and Methods 64 Amplification and cioning ofsfp ORF 64 Expression and purification of Sfp 64 Amplification and cioning of the luciferase ORF 65 Expression and purification of ybbr-luciferase 65 Coenzyme A-modified TMV-Lys 66 Covaient coupling of ybbr-luciferase to coenzyme A-modified TMV-Lys with Sfp in Solution 67 Covaient coupling of ybbr-luciferase to coenzyme A-modified TMV-Lys with Sfp on a solid Substrate 67 Electron microscopy 68 Results 69 Heterologous expression ofsfp in E. coli 69 Heterologous expression of ybbr-luciferase in E.coli 71 Coenzyme A modification of TMV-Lys 72 Covaient coupling of ybbr-luciferase to coenzyme A-modified TMV-Lys with Sfp in Suspension 74 Covaient coupling of ybbr-luciferase to coenzyme A-modified TMV-Lys with Sfp on a solid Substrate 75 Discussion 77 Acknowledg ements 79 4
Supplementary Data 80 References 82 TMV-based nanorods as a novel tool to gain insights into motor protein assem blies 87 Abstract 87 Introduction 88 Materials and Methods 91 Mutagenesis ofplasmldp843tmv-cys 91 Virus propagation and Isolation 92 RT-PCR of viral RNA and sequencing 93 Fluorescent labeling of TMV parthles 93 Isolation of motorprotein rk555-gfp 94 Microtubule afflnity purification 94 Modification of TMV-DM with maleimido-c3-nta and Atto 647N 95 SDS-PAGE 95 Enzyme-Iinked immunosorbent assay (ELISA) 95 Centrifugation assay Motility assay setup 96 96 Electron microscopy 97 Results 98 Construction ofplasmid p843tmv-dm 98 Infectivity and Symptoms 98 Particle Isolation 100 Verification offne mutations on the genomic level 100 Fluorescent labeling of TMV-DM particles 100 Motor protein rk555-gfp expression and purification 102 Double modification of TMV-DM particles with both Atto 647N and NTA 103 Enzyme-Iinked immunosorbent assay (ELISA) 104 Centrifugation assay Setup for a motility assay 105 106 5
Discussion 108 Acknowledgements 111 References 112 Gesamtliteraturverzeichnis 117 Danksagungen 129 Erklärung 131 Lebenslauf 133 6