Biochemical and functional characterization of cell-free expressed G-protein coupled receptors of the human endothelin system [Elektronische Ressource] / von Friederike Alessandra Junge

Friederike Ziegler

Sujets

• Biologie

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2010
• Nombre de lectures: 37
• Langue: Deutsch
• Poids de l'ouvrage: 9 Mo

Extrait

Biochemical and functional characterization of cell-free
expressed G-protein coupled receptors of the human
endothelin system











Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften



vorgelegt beim Fachbereich
Biochemie, Chemie und Pharmazie (FB 14)
der Goethe-Universität
in Frankfurt am Main








von
Friederike Alessandra Junge
aus Wiesbaden







Frankfurt 2010
(D30)



































Vom Fachbereich Biochemie, Chemie und Pharmazie (FB14)
der Goethe-Universität als Dissertation angenommen.


Dekan: Prof. Dr. Dieter Steinhilber

Gutachter: Prof. Dr. Volker Dötsch
PD Dr. Rupert Abele


Datum der Disputation:
















„Fantasie ist wichtiger als Wissen. Wissen ist begrenzt, Fantasie aber
umfasst die ganze Welt.“
Albert Einstein


































Table of contents I


Table of contents

Table of contents............................................................................................................. I
Abbreviations.................................................................................................................V
Summary .........................................................................................................................1
Zusammenfassung ..........................................................................................................4
1. Introduction..............................................................................................................10
1.1. Selecting expression systems for the large scale production of functional integral
membrane proteins.................................................................................................10
1.1.1. Basic characteristics of membrane proteins ......................................................... 10
1.1.2. Conventional cell-based expression of MPs 11
1.2. Cell-free expression of MPs ..................................................................................13
1.2.1. CF extract sources: Selection of the adequate expression environment............... 14
1.2.2. E. coli CF reaction compounds and major system configurations ....................... 15
1.2.3. CF reaction modes for the production of MPs...................................................... 18
1.3. G-protein coupled receptors – GPCRs ..................................................................21
1.3.1. Diversity, evolution and topology of GPCRs ........................................................ 21
1.3.2. Ligand binding and GPCR activation................................................................... 22
1.3.3. Biological membranes and their composition: Necessary lipids for MPs, especially
for GPCRs ............................................................................................................. 24
1.3.4. GPCR dimerization / oligomerization: An evaluation of current models ............. 25
1.3.5. Expression systems for the functional and structural characterization of GPCRs27
1.4. Structural and functional investigations on CF expressed MPs and GPCRs.........29
1.5. The endothelin system ...........................................................................................31
1.5.1. Evolution of the endothelin core system in the animal kingdom: Gene phylogeny
indicates its vertebrate specific origin .................................................................. 31
1.5.2. The human ET system: Receptors, ligands and binding kinetics .......................... 31
1.5.3. Physiology and pathophysiology of the human ET system.................................... 33
1.5.4. Lipid composition of mammalian ET receptor membranes: Requirement of
essential lipids?..................................................................................................... 35
1.5.5. Homo- and heterodimerization of ET receptors.................................................... 35
1.6. CF characterization of the human ETB receptor ...................................................36
1.7. Aim of this project.................................................................................................38
2. Materials...................................................................................................................39
2.1. Equipment..............................................................................................................39
2.2. Software.................................................................................................................40
2.3. Reagents and chemicals.........................................................................................40
2.4. Labelled chemicals and ligands.............................................................................43
2.5. Microbial strains ....................................................................................................43
2.6. Oligonucleotides43
2.7. Common buffers, media and reagents ...................................................................43
2.7.1. General buffers and media for the preparation of cell-free extract, T7 RNA
polymerase and bacterial growth.......................................................................... 43
2.7.2. Reagents and buffers for cell-free expression and mRNA preparation................. 45
2.7.3. Buffers for DNA-Agarose gels, SDS-PAGE and immunodetection by Western
Blotting.................................................................................................................. 46
2.7.4. Buffers for protein purification and protein analysis............................................ 47 II Table of contents

3. Methods.....................................................................................................................49
3.1. Standard methods of molecular biology and microbiology...................................49
3.1.1. DNA techniques: Conventional polymerase chain reaction (PCR) and cloning .. 49
3.1.2. Site-directed mutagenesis: Replacement of essential cysteines involved in disulfide
bridge formation.................................................................................................... 51
3.1.3. Template design for CF expression using E. coli S30 extracts ............................. 52
3.1.4. Basic working with E. coli: Transformation of plasmids, growth and storage..... 52
3.2. Preparation of T7 RNA polymerase for cell-free expression ................................53
3.3. Cell-free expression of MPs ..................................................................................54
3.3.1. Preparation of an individual E. coli S30 extract .................................................. 54
3.3.2. Continuous exchange cell-free configurations using E. coli extracts ................... 56
3.3.3. of an individual S30 wheat germ extract .......................................... 58
3.3.4. Preparation of mRNA templates for the cell-free translation in WGEs................ 58
3.3.5. Continuous exchange cell-free expression using WGE......................................... 59
3.4. Protein analysis......................................................................................................60
3.4.1. Electrophoresis and immunoblotting .................................................................... 60
3.4.2. Determination of protein concentration................................................................ 61
3.4.3. Acetone precipitation ............................................................................................ 62
3.5. Protein purification and analysis of sample homogeneity.....................................62
3.5.1. Resolubilization of MP precipitates ...................................................................... 62
3.5.2. Anion exchange chromatography: Purification of individually prepared T7RNAP
............................................................................................................................... 62
3.5.3. IMAC: Ni – and Co - NTA chromatography......................................................... 63
3.5.4. Streptavidin affinity ................................................................... 64
3.5.5. Size exclusion chromatography: A basic method to study homogeneity and stability 64
3.5.6. Multi angle light scattering: A more refined method for the calculation of protein
mass and its oligomerization state in detergent .................................................... 65
3.6. Affinity chromatography: Immobilization as a means to study receptor-receptor
interaction and ligand binding competence .....