Insertion of Cecropin A and reconstitution of bacterial outer membrane protein FhuA variants in polymeric membranes [Elektronische Ressource] / Noor Muhammad

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Insertion of Cecropin A and Reconstitution of Bacterial Outer Membrane Protein FhuA Variants in Polymeric Membranes Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigte Dissertation vorgelegt von Master of Biochemistry/Molecular Biology Noor Muhammad aus Bannu, Pakistan Berichter: Universitätsprofessor Dr. Ulrich Schwaneberg Universitätsprofessor Dr. Lothar Elling Tag der mündlichen Prüfung: 14.04.2011 Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar. DDDDDDContents ACKNOWLEDGEMENT ...........................................................................................................I List of Tables ............................................................................................................................III List of Figures .......................................................................................................................... IV List of Abbreviations ...............................................................................................................VII Abstract ....................................................................................................................................... 1 Chapter I-Introduction................................................................................

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• Biologie

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• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2011
• Nombre de lectures: 68
• Langue: Deutsch
• Poids de l'ouvrage: 7 Mo

Extrait

Insertion of Cecropin A and Reconstitution of
Bacterial Outer Membrane Protein FhuA
Variants in Polymeric Membranes

Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der
RWTH Aachen University zur Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften genehmigte Dissertation



vorgelegt von
Master of Biochemistry/Molecular Biology
Noor Muhammad
aus Bannu, Pakistan


Berichter: Universitätsprofessor Dr. Ulrich Schwaneberg
Universitätsprofessor Dr. Lothar Elling

Tag der mündlichen Prüfung: 14.04.2011


Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online
verfügbar.
Contents
ACKNOWLEDGEMENT ...........................................................................................................I
List of Tables ............................................................................................................................III
List of Figures .......................................................................................................................... IV
List of Abbreviations ...............................................................................................................VII
Abstract ....................................................................................................................................... 1
Chapter I-Introduction............................................................................................................... 3
1.1 Polymersome membrane functionalization with FhuA variant (a channel protein) .............3
1.1.2 Nanocompartments (General).................................................................................................................3
1.1.3 Mechanism of self assembly of block co-polymers into polymersomes.................................................4
1.1.4 Properties of polymer vesicles ................................................................................................................6
1.1.5 Polymersome applications ......................................................................................................................7
1.1.6 Protein-lipid and protein-polymer interactions .......................................................................................8
1.1.7 Functionalization of lipid and polymersome membranes .....................................................................11
1.1.7.1 Wild type FhuA Protein.....................................................................................................................14
1.1.7.2 Concept of the FhuA variant, FhuA 1-159 Extended ......................................................................15
1.2 Polymersome membrane functionalization with the peptide-protein chimera Cecropin A-
EGFP 17
1.2.1 Cecropin A............................................................................................................................................17
1.2.2 Behavior of Cecropin A in contact with the lipid membrane................................................................17
1.2.3 Hydrophobic peptides and polymersomes ............................................................................................20
1.2.4 Polymersome surface functionalization and targeting ..........................................................................20
1.2.5 GFP and EGFP (enhanced green fluorescence protein)........................................................................22
1.2.6 Aim of the study ...................................................................................................................................24
Chapter II: Materials and Methods......................................................................................... 25
2.1 FhuA 1-159 Ext. cloning, expression, extraction and its reconstitution in the
polymeric membranes 25
2.1.1 Bacterial strains and media used for culturing, cloning and expression of FhuA 1-159 Ext..............25
2.1.2 Chemicals and synthetic gene...............................................................................................................26
2.1.3 Cloning of the FhuA 1-159 Ext..........................................................................................................26
2.1.4 FhuA 1-159 Ext transformation for expression..................................................................................27
2.1.5 Expression of FhuA 1-159 Ext...........................................................................................................27
2.1.6 Extraction of FhuA 1-159 Ext ............................................................................................................28
2.1.6.1 Organic solvent extraction of FhuA 1-159 Ext from the membrane pellet .....................................29
2.1.7 SDS-PAGE ...........................................................................................................................................30
2.1.8 Secondary structure prediction of FhuA variants..................................................................................31
2.1.9 Circular dichroism for FhuA 1-159 Ext .............................................................................................31
2.1.10 Properties of polymer PIB -PEG -PIB ...................................................................................31 1000 6000 1000
2.1.11 Molecular Dynamics PIB -PEG -PIB ....................................................................................32 1000 1500 1000
2.1.11.1 System assembly..............................................................................................................................33
2.1.12 Size exclusion chromatography ..........................................................................................................34
2.1.13 Encapsulation of HRP and insertion of FhuA 1-159 Ext as channel protein into polymersomes
using PIB -PEG -PIB polymer .........................................................................................................34 1000 6000 1000
2.1.14 TMB assay for HRP............................................................................................................................35
2.1.15 Biotinylation of Lysine residues in the FhuA 1-159 Ext channel protein inserted into polymersomes
.......................................................................................................................................................................35
2.1.16 Approximation of the number of biotin labels....................................................................................36


DDDDDD2.2 Cloning, expression and purification of Cecropin A-EGFP chimera, EGFP and
polymersome membrane functionalization with Cecropin A-EGFP ......................................... 37
2.2.1 Bacterial strains and media used for culturing, cloning and expression of Cecropin A-EGFP and
EGFP .............................................................................................................................................................37
2.2.2 Cloning of Cecropin A-EGFP-C-His chimera ......................................................................................38
2.2.3 Expression of pALXtreme-3b–Cecropin A- EGFP–C-His...................................................................39
2.2.4 Expression of EGFP-C-His...................................................................................................................40
2.2.5 Extraction and purification of Cecropin A-EGFP and EGFP ...............................................................40
2.2.6 Concentration measurement of the protein and SDS-PAGE.................................................................41
2.2.7 Preparation of polymersomes from PIB -PEG -PIB polymer and insertion of Cecropin A-1000 6000 1000
EGFP .............................................................................................................................................................41
2.2.8 DLS (dynamic light scattering).............................................................................................................42
2.2.9 Tryptophane fluorescence emission shift assay ....................................................................................43
2.2.10 Circular dichroism (CD) measurement ...............................................................................................43
2.2.11 EGFP denaturation and renaturation...................................................................................................44
2.2.12 Cecropin A-EGFP denaturation and pepsin digestion on the polymersome surface.........................44
2.2.13 Calcein leakage experiment ................................................................................................................45
Chapter III-Results................................................................................................................... 46
3.1 Rational design of FhuA 1-159 Ext.......................................................................................................46
3.2 Cloning expression and purification of FhuA ∆1-159 Ext and its reconstitution in polymeric
membranes 48
3.2.1 Cloning ...............................................................................................................