Molecular recognition in BMP ligand-receptor interactions [Elektronische Ressource] / vorgelegt von Stefan Harth

Stefan Harth

Sujets

• Biologie

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2010
• Nombre de lectures: 27
• Langue: English
• Poids de l'ouvrage: 17 Mo

Extrait

MOLECULAR RECOGNITION IN
BMP LIGAND-RECEPTOR INTERACTIONS


DISSERTATION ZUR ERLANGUNG DES
NATURWISSENSCHAFTLICHEN DOKTORGRADES
DER JULIUS-MAXIMILIANS-UNIVERSITÄT WÜRZBURG

VORGELEGT VON

STEFAN HARTH

AUS BAD NEUSTADT AN DER SAALE







WÜRZBURG, SEPTEMBER 2010











Eingereicht am: .........................................................................




Mitglieder der Promotionskommission:


Vorsitzender: .........................................................................

1. Gutachter: Prof. Dr. Walter Sebald

2. Gutachter: Prof. Dr. Thomas Müller



Tag des Promotionskolloquiums: .........................................................................



Doktorurkunde ausgehändigt am: .........................................................................











MEINEN ELTERN

























Table of contents
1. Summary..............................................................................................................................1
Zusammenfassung ...............................................................................................................3
2. Introduction .........................................................................................................................5
2.1. BMPs and other ligands of the TGF- superfamily ....................................................5
2.2. Receptors of the TGF- superfamily...........................................................................9
2.3. Signal transduction in the TGF- superfamily ..........................................................11
2.4. Ligand receptor interaction: promiscuity and specificity..........................................12
2.5. Ligand-receptor interplay in the case of heterodimeric BMP-ligands ......................15
2.6. Structural aspects of ligand-receptor interaction using the example of BMPR-IA..17
2.7. Aim of the study ........................................................................................................19
3. Materials and methods.......................................................................................................20
3.1. List of abbreviations ..................................................................................................20
3.2. Materials ....................................................................................................................22
3.2.1. Antibodies..........................................................................................................22
3.2.2. Bacterial strains .................................................................................................22
3.2.3. Cell lines............................................................................................................23
3.2.4. Chemicals23
3.2.5. Growth factors ...................................................................................................23
3.2.6. Kits ....................................................................................................................24
3.2.7. Oligonucleotides................................................................................................24
3.2.8. Plasmids.............................................................................................................25
3.2.9. Restriction enzymes...........................................................................................25
3.2.10. Sterile working tools..........................................................................................26
3.3. Molecular biological methods ...................................................................................26
3.3.1. Generation of BMP ligand and BMP receptor expression constructs...............26
3.3.2. Mutagenesis of BMPR-IA .............................................................................27 ecd
3.3.3. Agarose gel electrophoresis...............................................................................28
3.4. Protein chemical methods29
3.4.1. Polyacrylamide gel electrophoresis (SDS-PAGE) ............................................29
3.4.2. Protein staining..................................................................................................30
3.4.3. Molecular weight standards for SDS-PAGE.....................................................32
3.4.4. Photometric determination of protein concentrations .......................................32
3.4.5. Determination of protein concentrations using bicinchoninic acid assay .........33
3.4.6. Mass spectrometry.............................................................................................34
I Table of contents
3.4.7. Western blotting ................................................................................................34
3.5. Expression of recombinant proteins in E.coli............................................................36
3.5.1. Culture medium .................................................................................................36
3.5.2. Storage of E.coli cultures...................................................................................37
3.5.3. Generation of competent E.coli cells.................................................................38
3.5.4. Transformation of competent E.coli cells..........................................................38
3.5.5. Expression of BMP ligands ...............................................................................38
3.5.6. Expression of BMP receptors............................................................................39
3.6. Purification of recombinant proteins .........................................................................40
3.6.1. Purification of BMP ligands..............................................................................40
3.6.2. Purification of BMP receptors...........................................................................44
3.6.3. Reverse phase high performance liquid chromatography (RP-HPLC) .............49
3.6.4. Gel filtration chromatography50
3.7. Biosensor analysis using the BIAcore technique ......................................................51
3.8. Preparation and crystallization of antibody-receptor complexes ..............................52
3.9. X-ray diffractometry..................................................................................................52
3.9.1. Measurement of X-ray diffraction data .............................................................52
3.9.2. Processing and analysis of X-ray diffraction data.............................................53
3.10. Determination of protein activity in eukaryotic cell-based assays............................53
3.10.1. General handling of cell cultures and overview of culture media.....................53
3.10.2. Cell counting via trypan blue staining...............................................................54
3.10.3. Alkaline phosphatase assay ...............................................................................54
3.10.4. Smad phosphorylation assay .............................................................................55
33.10.5. H-Thymidine incorporation assay....................................................................56
3.10.6. IH-1 apoptosis assay..........................................................................................56
4. Expression, biochemical characterization and biological analysis of BMP heterodimers 57
4.1. Expression and purification of BMP ligands.............................................................57
4.1.1. BMP-2 ...............................................................................................................57
4.1.2. BMP-2/6 ............................................................................................................59
4.1.3. BMP2/7..............................................................................................................63
4.1.4. Summary of BMP ligand generation.................................................................69
4.2. BIAcore interaction analysis of ligand receptor interplay.........................................70
4.3. Biological activity in cell assays ...............................................................................72
4.3.1. Alkaline phosphatase assay72
II Table of contents
34.3.2. H-Thymidine incorporation assay....................................................................78
4.3.3. IH-1 apoptosis assay..........................................................................................80
4.3.4. Smad phosphorylation assay .............................................................................82
5. Structural and functional analysis of an antibody/BMPR- IA complex ........................84 ecd
5.1. Preparation of BMPR-IA wildtype and mutants....................................................84 ecd
5.2. Complex preparation