The human GPCR nicotinic acid receptor 1 [Elektronische Ressource] : heterologous overproduction in Pichia pastoris and the reconstitution of its complex with β-Arrestin 1 in vivo and in vitro / von Jan Griesbach

Jan.W

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• Gesundheit

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2007
• Nombre de lectures: 8
• Langue: English
• Poids de l'ouvrage: 15 Mo

Extrait

The human GPCR Nicotinic Acid Receptor 1:
Heterologous Overproduction in Pichia pastoris and
the Reconstitution of its Complex with β-Arrestin 1
in vivo and in vitro




Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften


vorgelegt beim Fachbereich
Chemische und Pharmazeutische Wissenschaften
der Johann Wolfgang Goethe Universität
Frankfurt am Main


von
Jan Griesbach
aus Augsburg, Deutschland


Frankfurt am Main, 2007
D30
1













vom Fachbereich Chemische und Pharmazeutische Wissenschaften der Johann Wolfgang
Goethe – Universität als Dissertation angenommen














Dekan: Prof. Dr. Harald Schwalbe
1. Gutachter: Prof. Dr. Clemens Glaubitz
2. rof. Dr. Hartmut Michel
Datum der Disputation:
2


3



















As I stand here, the ground beneath is nothing more than one point of view
(Dave Matthews – Raven)


4 Table of Content
Table of Content

Abstract ............................................................................... 8
Zusammenfassung ............................................................ 10
1 Introduction...................................................................15
1.1 Signal Transduction..................................................................................................15
1.1.1 GPCRs..............................................................................................................16
1.1.2 GPCR Signaling...............................................................................................23
1.1.3 Dimerization.....................................................................................................29
1.1.4 GPCRs as Drug Targets ................................................................................... 30
1.2 Crystallization of Membrane Proteins...................................................................... 31
1.3 Nicotinic Acid Receptor........................................................................................... 33
1.3.1 Nicotinic Acid in Clinical Use ......................................................................... 33
1.3.2 Discovery of the Nicotinic Acid Receptor ....................................................... 35
1.3.3 Nicotinic Acid Receptor Drugs ........................................................................ 35
1.3.4 Nicotinr Expression and Signaling......................................... 35
1.3.5 Physiological Ligand........................................................................................36
1.4 Aim of the Project .................................................................................................... 37
2 Material & Methods....................................................... 38
2.1 Chemicals.................................................................................................................38
2.1.1 General Chemicals...........................................................................................38
2.1.2 Labeled Chemicals40
2.1.3 Detergents.........................................................................................................40
2.1.4 Columns, Chromatographic Matrices & Prepacked Columns ......................... 41
2.1.5 Antibodies........................................................................................................42
2.1.6 Enzymes...........................................................................................................43
2.1.7 Kits & Markers................................................................................................. 43
2.1.8 Filters, Membranes & Concentrators ............................................................... 44
2.1.9 Buffers & Solutions.......................................................................................... 44
2.1.10 Vectors.............................................................................................................49
2.1.11 cDNA-Templates.............................................................................................49
2.1.12 Primers49
5Table of Content
2.1.13 Strains...............................................................................................................52
2.1.14 Media................................................................................................................53
2.1.15 General Apparatus General .............................................................................. 58
2.2 Methods.................................................................................................................... 59
2.2.1 Genetic Engineering.........................................................................................59
2.2.2 Transformation of P. pastoris .......................................................................... 65
2.2.3 Protein Expression............................................................................................67
2.2.4 Cytosol & Membrane Preparation.................................................................... 70
2.2.5 Radioligand Binding Assay.............................................................................. 71
2.2.6 Solubilization...................................................................................................75
2.2.7 Protein Purification..........................................................................................76
2.2.8 Stability Screen................................................................................................80
2.2.9 Activity Measurements....................................................................................82
2.2.10 Reconstitution85
2.2.11 Electron Microscopic Imaging ......................................................................... 87
2.2.12 NMR Spectroscopic Measurements ................................................................. 88
2.2.13 Mammalian Cell Culture.................................................................................. 90
2.2.14 Interaction of β-Arrestin 1-382 with NAR1..................................................... 91
2.2.15 General Techniques..........................................................................................91
3 Results..........................................................................95
3.1 Production of the human GPCR HM74A in P. pastoris........................................... 95
3.1.1 Cloning.............................................................................................................95
3.1.2 Transformation & Multi-Copy Clone Selection............................................... 97
3.1.3 Expression Analysis.........................................................................................98
3.1.4 Expression Optimization................................................................................103
3.1.5 Effect of Various Buffer Components: cations, pH, imidazole, DMSO and
DTT 107
3.1.6 Solubilization.................................................................................................109
3.1.7 Purification & Enzymatic Processing ............................................................ 113
3.1.8 Stability Screen..............................................................................................117
3.1.9 Chromatographic Purification of NAR1 (with tags)...................................... 119
3.2 Interaction of NAR1 with β-Arrestin 1 in vivo...................................................... 124
3.3 Comparative Multi-Host Expression of β-Arrestins in E. coli and P. pastoris ...... 126
3.3.1 Cloning & Expression .................................................................................... 126
6 Table of Content
3.3.2 Purification.....................................................................................................129
3.4 Interaction of NAR1 with β-Arrestin 1 in vitro...................................................... 131
3.5 Activity Measurements of Solubilized & Purified NAR1 ..................................... 132
3.6 Reconstitution of NAR1 into Liposomes ............................................................... 135
3.7 NMR Measurements of β-Arrestin 1-382 .............................................................. 137
4 Discussion..................................................................140
4.1 Production of the human GPCR HM74A in P. pastoris......................................... 140
4.1.1 Multi-Copy Clone Selection...........................................................................141
4.1.2 Pharmacological Characterization142
4.1.3 Expression Optimization of NAR1 ................................................................ 143
4.1.4 Solubilization.................................................................................................145
4.1.5 Purification...........................................................