Structural and functional analysis of virulence-associated listerial surface proteins [Elektronische Ressource] / von Maike Bublitz

Mbu

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Informations

• Publié par: technische_universitat_carolo-wilhelmina_zu_braunschweig
• Publié le: 01 janvier 2008
• Nombre de lectures: 19
• Langue: Deutsch
• Poids de l'ouvrage: 15 Mo

Extrait

Structural and Functional Analysis
of Virulence-Associated
Listerial Surface Proteins






Von der Fakultät für Lebenswissenschaften

der Technischen Universität Carolo-Wilhelmina

zu Braunschweig

zur Erlangung des Grades einer

Doktorin der Naturwissenschaften

(Dr. rer. nat.)

genehmigte

D i s s e r t a t i o n












von Maike Bublitz
aus Wolfenbüttel






















1. Referent: Honorarprofessor Dr. Dirk Heinz
2. Referent: Professor Dr. Michael Steinert
eingereicht am: 05.11.2007
mündliche Prüfung (Disputation) am: 20.12.2007

Druckjahr 2008

Vorveröffentlichungen der Dissertation

Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät für
Lebenswissenschaften, vertreten durch den Mentor der Arbeit, in folgenden Beiträgen vorab
veröffentlicht:


Tagungsbeiträge
Bublitz, M., Heinz, D. W. & Schubert, W. D.: Structural Basis for Autoinhibition of an
Autolysin involved in Listeria monocytogenes Pathogenicity. (Poster), Murnau Conference
on Structural Biology of Disease Mechanisms, Murnau (2007).

Bublitz, M., Holland, C., Heinz, D. W. & Schubert, W. D.: Crystal Structure of InlJ’: A
Leucine-Rich Repeat Protein with a Cysteine Ladder. (Poster), Jahrestagung der Deutschen
Gesellschaft für Kristallographie, Bremen (2007). Poster-Preis.

Bublitz, M., Heinz, D. W. & Schubert, W. D.: Peptidoglycan and Pathogenesis –
thCharacterization of a Listerial Autolysin. (Vortrag) 9 Heart of Europe Bio-Crystallography
Meeting, Teistungenburg (2006).

Bublitz, M., Heinz, D. W. & Schubert, W. D.: Structure and Function of an Autolysin
thinvolved in the Pathogenicity of Listeria monocytogenes. (Poster) 8 International School
on the Crystallography of Biological Macromolecules, Como, Italien und Jahrestagung der
Deutschen Gesellschaft für Kristallographie, Freiburg (2006).

Bublitz, M., Stradal, T., Heinz, D. W. & Schubert, W. D.: The Internalin Family of Listerial
Surface Proteins: Analysis of LRR-Proteins involved in Infection. (Poster) Murnau
Conference on Structural Biology of Molecular Recognition, Murnau (2005).


















Für Benno Laumert,
der viel zu früh das getan hat, was nur die Besten tun.







Contents
Abbreviations ........................................................................................................................ 1
Summary................................................................................................................................ 4
1 Introduction................................................................................................................... 6
1.1 Listeria monocytogenes and listeriosis ....................................................................... 7
1.2 Surface-located and secreted virulence factors mediate intracellular infection.......... 9
1.3 Autolysins................................................................................................................. 11
1.3.1 The Gram-positive cell wall ........................................................................... 11
1.3.2 Autolysins in L. monocytogenes..................................................................... 13
1.3.3 The autolysin Auto ......................................................................................... 13
1.4 Internalins: Surface-located leucine-rich repeat proteins ......................................... 14
1.4.1 InlJ..................................................................................................................16
1.4.2 InlG17
2 Aims and Scope ........................................................................................................... 18
2.1 The autolysin Auto.................................................................................................... 18
2.2 Internalin family proteins.......................................................................................... 18
3 Material and Methods ................................................................................................ 20
3.1 Standard Materials20
3.1.1 Chemicals, enzymes and kits.......................................................................... 20
3.1.2 Molecular weight standards............................................................................ 20
3.1.3 Crystallization screens....................................................................................21
3.1.4 Bacterial strains..............................................................................................21
3.1.5 Plasmids..........................................................................................................22
3.1.6 Oligonucleotides.............................................................................................23
3.1.7 Media..............................................................................................................25
3.1.8 Antibiotics......................................................................................................26
3.2 Microbiology.............................................................................................................26
3.2.1 Agar plates26
i 3.2.2 Liquid culture..................................................................................................26
3.2.3 Long-term storage...........................................................................................26
3.2.4 Isolation of peptidoglycan from Listeria innocua .......................................... 27
3.3 Molecular biology.....................................................................................................28
3.3.1 Preparation of electrocompetent cells............................................................. 28
3.3.2 Preparation of chemically competent cells..................................................... 28
3.3.3 Transformation of competent bacteria............................................................ 28
3.3.4 Plasmid preparation........................................................................................29
3.3.5 Determining DNA concentration and purity .................................................. 29
3.3.6 Agarose gel electrophoresis............................................................................30
3.3.7 Extraction of DNA from agarose gels 30
3.3.8 Digestion of plasmid DNA with restriction endonucleases............................ 30
3.3.9 Dephosphorylation of linearised plasmid DNA ............................................. 30
3.3.10 Ligation of DNA fragments30
3.3.11 Polymerase chain reaction .............................................................................. 31
3.3.12 Design and synthesis of deoxyribo-oligonucleotides ..................................... 31
3.3.13 Site-directed mutagenesis...............................................................................32
3.3.14 DNA sequencing.............................................................................................32
3.4 Protein production and purification .......................................................................... 32
3.4.1 Test expressions..............................................................................................32
3.4.2 Recombinant protein synthesis....................................................................... 33
3.4.3 Production of selenomethionine-labelled protein........................................... 33
3.4.4 Cell lysis.........................................................................................................34
3.4.5 Affinity chromatography................................................................................34
3.4.6 Proteolytic cleavage of GST-fusion proteins.................................................. 35
3.4.7 Dialysis...........................................................................................................35
3.4.8 Ion exchange chromatography........................................................................ 35
3.4.9 Gel permeation chromatography .................................................................... 36
3.4.10 Concentration of protein solutions.................................................................. 36
3.5 Protein biochemical methods....................................................................................37
3.5.1 Photometric quantification of protein concentration ...................................... 37
3.5.2 SDS-polyacrylamide gel electrophoresis (SDS-PAGE)................................. 37
3.5.3 Transfer of proteins to membranes (Western Blot) ........................................ 38
ii 3.5.4 Immunodetection of immobilized proteins..................................................... 39
3.5.5 N-terminal sequencing...........................