Overexpression and purification of membrane proteins in yeast [Elektronische Ressource] : the GPCR α-factor receptor and a methanococcal transporter / Anna Le Bris

Lebris Anna

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 18
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades

der Fakultät für Chemie und Pharmazie

der Ludwig-Maximilians-Universität München










Overexpression and purification of membrane proteins in yeast:
The GPCR α-factor receptor and a methanococcal transporter












Anna Le Bris


aus


Quimper (Frankreich)

















2007
12

Erklärung

Diese Dissertation wurde im Sinne von §13 Abs.3 bzw. 4 der Promotionsordnung vom
29. Januar 1998 von Herrn Prof. Dr. Dieter Oesterhelt betreut.















Ehrenwörtliche Versicherung

Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.


München, am 27. April 2007






















Dissertation eingereicht am 27te April 2007

1. Gutachter Prof. Dr. D.Oesterhelt
2. Gutachter Prof. Dr. R. Beckmann


Mündliche Prüfung am 19. Oktober 2007
34



Communications




Publication in preparation:


Anna Le Bris, Birgit Wiltschi, Douglas D. Griffith, Dieter Oesterhelt.
Expression and purification of a homogeneous functional Saccharomyces cerevisiae
α-factor receptor variant




Poster presentation:

Anna Le Bris, Douglas D. Griffith, Dieter Oesterhelt

Heterologous overexpression and purification of a putative amino-acid transporter from
Methanococcus jannaschii

rd3 international conference on structure, dynamics and function of proteins in biological
membranes, Switzerland, Ascona, 14 -19 May 2006































5


























































6


7Contents
Contents

Contents................................................................................................................................................1

Summary...............................................................................................................................................7

Introduction ...................................................................................................................9
1. Structure and function of membrane proteins: challenges of the study ............................9
1.1. Importance of membrane proteins .........................................................................................9
1.2. The difficulties in studying membrane proteins......................................................................9
2. Membrane protein families of interest..................................................................................11
2.1. G-Protein Coupled Receptors..............................................................................................11
2.2. Hyperthermophilic transporters from Methanococcus jannaschii.........................................14
2.2.1. The archeon Methanococcus jannaschii (Methanocaldococcus jannaschii)...................14
2.2.2. Hyperthermophilic proteins from M.jannaschii.................................................................14
3. Characteristics of the proteins of interest ...........................................................................16
3.1. Ste2p, a G-Protein Coupled Receptor of Saccharomyces cerevisiae..................................16
3.1.1. Function of Ste2p ............................................................................................................16
3.1.2. Secondary structure of Ste2p..........................................................................................18
3.2. Hyperthermophilic transporters from M.jannaschii...............................................................19
4. Reasons for the overexpression of Ste2p and hyperthermophilic transporters in
S.cerevisiae .............................................................................................................................20
4.1. Expression of the GPCR Ste2p in S.cerevisiae ...................................................................20
4.2. Expression of the hyperthermophilic membrane proteins in S.cerevisiae............................21

Results ................................................................................................................................................25
5. Homologous overexpression and isolation of the Saccharomyces cerevisiae α-factor
receptor Ste2p in Saccharomyces cerevisiae......................................................................25
5.1. Cloning strategy ...................................................................................................................25
5.1.1. Ste2p amino-acid sequence alterations: mutations of N-glycosylations sites and
truncation of the C-terminal part......................................................................................25
m5.1.2. Construction of the Ste2 p expression vector pT326 and the expression strain YpT326
........................................................................................................................................26
5.1.3. Alternative expression strains..........................................................................................26
m5.2. Selection of a suitable Ste2 p expression strain .................................................................27
m5.2.1. Selection by Ste2 p expression level..............................................................................27
5.2.2. Alpha-factor functionality test I: the halo assay ...............................................................28
5.2.3. functionality test II: the binding-assay, characterisation of binding properties
K /B .............................................................................................................................30 d max
5.2.4. Alpha-factor functionality test III: competitive inhibition assays, stereospecifity..............30
15.3. Culture and overexpression .................................................................................................31
m5.4. Solubilisation of Ste2 p........................................................................................................34
5.4.1. ion ...................................................................................................................34
5.4.2. Presolubilisation ..............................................................................................................36
5.5. Purification ...........................................................................................................................39
5.5.1. One-step purification .......................................................................................................39
5.5.1.1. Metal-affinity chromatography, with Ni2+-NTA beads ..............................................39
5.5.1.1.1. Beads volume.....................................................................................................41
5.5.1.1.2. β-mercaptoethanol and EDTA concentration .....................................................41
5.5.1.1.3. Urea....................................................................................................................42
5.5.1.1.4. Deglycosylation...................................................................................................43
2+ 2+ +5.5.1.2. Other metal-affinity purifications: Cu -NTA, Co -NTA, Zn -NTA............................43
5.5.2. Two-step purification45
5.5.2.1. FLAG-affinity chromatography..................................................................................45
5.5.2.2. Anion-exchange chromatography.............................................................................46
5.5.2.3. Size-exclusion chromatography ...............................................................................48
5.5.3. Final purification protocol ................................................................................................49
m5.6. Characterisation of Ste2 p...................................................................................................50
5.6.1. N-terminal sequencing analysis.......................................................................................50
5.6.2. MALDI-TOF mass spectrometric analysis............................................................