Crystallisation and functional studies with ethylene receptor ETR1 [Elektronische Ressource] / Elisa Buchen. Gutachter: Dieter Willbold. Betreuer: Georg Groth

Ellie Gunesch

Sujets

• Naturwissenschaften
• Biologie

Informations

• Publié par: heinrich-heine-universitat_dusseldorf
• Publié le: 01 janvier 2011
• Nombre de lectures: 68
• Langue: Deutsch
• Poids de l'ouvrage: 11 Mo

Extrait

Crystallisation and functional studies
with ethylene receptor ETR1
Inaugural-Dissertation
Elisa Buchen






Crystallisation and functional studies
with ethylene receptor ETR1







Inaugural-Dissertation


zur Erlangung des Doktorgrades
der Mathematisch-Naturwissenschaftlichen Fakultät
der Heinrich-Heine-Universität Düsseldorf









vorgelegt von


Elisa Buchen
aus Remscheid




Düsseldorf, Februar 2011


Aus dem Institut für Biochemie der Pflanzen
der Heinrich-Heine-Universität Düsseldorf



















Gedruckt mit der Genehmigung der
Mathematisch-Naturwissenschaftlichen Fakultät der
Heinrich-Heine-Universität Düsseldorf



Referent: Prof. Dr. G. Groth
Koreferent: Prof. Dr. D. Willbold

Tag der mündlichen Prüfung: 14.04.2011


Titelbild: Arabidopsis thaliana Ökotyp Landsberg erecta. Die Pflanze wurde freundlicherweise
zur Verfügung gestellt von Helge Pallakies, Institut für Genetik der Universität Düsseldorf.

Contents
1 Abstract..........................................................................................................7
2 Zusammenfassung...........................................................................................9
3 Introduction.................................................................................................. 11
3.1 Plant hormones: Communication is all!.................................................................................11
3.2 The plant hormone ethylene.................................................................................................11
3.2.1 The role of ethylene in plant life cycle....................................................................12
3.2.2 Ethylene biosynthesis.............................................................................................12
3.2.3 The ethylene signalling pathway in Arabidopsis thaliana ........................................13
3.2.4 Cross-talk between ethylene and other plant hormones...........................................19
3.2.5 Objectives of this thesis..........................................................................................20
4 Materials & Methods.................................................................................... 21
4.1 Materials..............................................................................................................................21
4.1.1 Equipment .............................................................................................................21
4.1.2 Chromatography supplies.......................................................................................22
4.1.3 Crystallographic supplies........................................................................................22
4.1.4 Kits........................................................................................................................23
4.1.5 Filters and Membranes...........................................................................................23
4.1.6 Chemicals and Buffers............................................................................................23
4.1.7 Radiochemicals.......................................................................................................24
4.1.8 Enzymes ................................................................................................................24
4.1.9 Antibodies..............................................................................................................25
4.1.10 Oligonucleotides.....................................................................................................25
4.1.11 Custom gene synthesis ...........................................................................................27
4.1.12 Vectors ..................................................................................................................27
4.1.13 Bacterial strains: Escherichia coli...........................................................................29
4.2 Microbiological methods .......................................................................................................30
4.2.1 Media for cultivation of E. coli cells.......................................................................30
4.2.2 Preparation and transformation of chemically competent E. coli cells.....................30
4.3 Molecular biological methods................................................................................................31
4.3.1 Amplification and isolation of plasmid DNA from E. coli .......................................31
4.3.2 Determination of DNA concentration .....................................................................31
4.3.3 Polymerase chain reaction to amplify DNA ............................................................31
4.3.4 Agarose gel electrophoresis.....................................................................................32
4.3.5 Cloning, Mutagenesis and Sequencing.....................................................................33
4.3.6 SLIC: Sequence and ligation independent cloning ...................................................34
4.4 Protein biochemical methods................................................................................................35
4.4.1 Protein quantification.............................................................................................35
4.4.2 SDS polyacrylamide gel electrophoresis...................................................................36
4.4.3 Silver staining of SDS-PAGE gels...........................................................................36
4.4.4 Western blotting ....................................................................................................37
3
4.4.5 Dot blotting...........................................................................................................37
4.4.6 Immunodetection ...................................................................................................38
4.4.7 Proteolytic cleavage ...............................................................................................38
4.5 Expression of recombinant proteins in E. coli .......................................................................39
4.5.1 Expression of full-length receptor protein AtETR1.................................................39
4.5.2 Expression of the extramembrane domain of AtETR1 ............................................40
4.5.3 Expression of PpETR1 and LeETR1......................................................................40
4.6 Purification of recombinant proteins from E. coli – general methods.....................................41
4.6.1 Cell disruption .......................................................................................................41
4.6.2 Isolation and solubilisation of E. coli membranes....................................................41
4.6.3 Isolation and purification of inclusion bodies ..........................................................42
4.6.4 Solubilisation of inclusion bodies ............................................................................42
4.6.5 Refolding of proteins solubilised from inclusion bodies............................................43
4.6.6 Size exclusion chromatography...............................................................................43
4.6.7 Removal of DnaK contamination............................................................................44
4.7 Purification protocols for individual proteins ........................................................................45
4.7.1 Purification of full-length receptor protein AtETR1................................................45
4.7.2 Purification of the extramembrane domain of AtETR1...........................................47
4.7.3 Purification of full-length PpETR1 and LeETR1....................................................48
4.7.4 Preparation of truncated PpETR1 and LeETR1 from inclusion bodies ...................48
4.8 Protein characterisation........................................................................................................49
4.8.1 In vitro kinase assay...............................................................................................49
4.8.2 Circular dichroism spectroscopy .............................................................................50
4.8.3 Fluorescence spectroscopy......................................................................................50
314.8.4 P-NMR spectroscopy............................................................................................51
4.8.5 Protein crystallography..........................................................................................52
4.8.6 Bioinformatics methods and software tools.............................................................57
5 Part A: Functional studies on ethylene receptor ETR1 from A. thaliana.. 59
5.1 Intrinsic tryptophan