Semisynthesis of light switchable human STAT6 variants and their characterization [Elektronische Ressource] / Sunanda Lahiri

Sujets

• Biologie

Informations

• Publié par: technischen_universitat_dortmund
• Publié le: 01 janvier 2008
• Nombre de lectures: 18
• Langue: English
• Poids de l'ouvrage: 8 Mo

Extrait

Semisynthesis of light switchable
human STAT6 variants and their
characterization

Dissertation

zur Erlangung des akademischen Grades
eines Doktors der Naturwissenschaften
(Dr. rer. nat.)
des Fachbereichs Chemie der TU Dortmund
Angefertigt am Max-Planck-Institut für molekulare Physiologie
in Dortmund (Germany)






Sunanda Lahiri
aus Mumbai (Indien)










Mom, Dad
&
Sis


Erklärung/ Declaration

Die vorliegende Arbeit wurde am Max-Planck-Institut für molekulare Physiologie,
Dortmund, Germany in der Abteilung Physikalische Biochemie von Prof. Dr. Roger S.
Goody, unter Anleitung von Prof. Dr. Christian Becker und Prof. Dr. Martin Engelhard
durchgeführt. Der experimentelle Teil wurde vom September 2004 bis November 2008
angefertigt. Hermit versichere ich an Eides statt, dass ich die vorliegende Arbeit
selbständig und nur mit den angegebenen Hilfsmitteln angerfertigt habe.


The present work was accomplished in the Max-Planck Institute for Molecular
Physiology, Dortmund, Germany in the department of Prof. Dr. Roger S. Goody under
the supervision of Prof. Dr. Christian Becker and Prof. Dr. Martin Engelhard. I hereby
declare that I performed the work presented independently and did not use any other
but the indicated aids.






1. Berichterstatter : Prof. Dr. M. Engelhard
2. Berichterstatter : Prof. Dr. Peter Eilbracht




Dortmund, November 2008 Sunanda Lahiri




Wisdom begins with
wonder
Socrates (469 BC – 399 BC)
Contents

Introduction

1.1. Overview 1
1.1.1 STATs 1
1.1.2 Structure of STATs 3
1.1.3 Activation mechanism of STATs 6
1.1.4 STAT6 10

1.2. Chemical protein synthesis 11
1.2.1. Native chemical ligation 12
1.2.2. Expressed protein ligation 16
1.2.3. Caged compounds 19

Aim

2.1. Aim of the project 22

Materials and methods

3.1. Materials and instruments 24
3.1.1 Materials 24
3.1.2 Instruments and suppliers 25
3.2. Chemical Methods 26
3.2.1 Synthesis of caged phosphorylated tyrosine building block 26
3.2.2 1-(2-nitrophenyl) ethanol (NPE- OH) (2) 26
3.2.3 O-1-(2-nitrophenyl) ethyl-O’-β-cyanoethyl-N,N-diisopropyl
phosphoramidite (3) 27
3.2.4 N-α-Fmoc-L-tyrosine tert- butyl Ester (5) 28
3.2.5 N-α-Fmoc-phospho (1-nitrophenylethyl-2-cyanoethyl)-
L-tyrosine tert-butyl ester. (7) 29
3.2.6 N-α-Fmoc-phospho (1-nitrophenylethyl-2-cyanoethyl)-L-tyrosine (8) 30
3.2.7 Synthesis of Cy5-NHS and coupling to the ε-amino group of Lysine 31 3.2.8 Synthesis of cysteine capture beads 32
3.2.9 Peptide synthesis 32
3.2.10 Cleavage of peptides attached to Wang resin 33
3.2.11 Peptide purification 33

3.3. Biochemical methods 34
3.3.1 Buffers and Media 34
3.3.2 Expression of recombinant STAT6 (aa 1 - 634) α-COSR in E. coli 35
3.3.3 Purification of STAT6MxeCBD 35
3.3.4 Determination of Protein Concentration 36

3.4. Analytical methods 36
3.4.1 Electro Spray Ionisation (ESI) - Mass spectrometry 36
3.4.2 Matrix-assisted laser desorption ionization (MALDI) 36
3.4.3 Nuclear Magnetic Resonance Spectroscopy 37
3.4.4 Thin-Layer Chromatography (TLC) 37
3.4.5 Sodium dodecyl sulfate polyacrylamide gel electrophoresis 38
3.4.6 Silver staining 39
3.4.7 Non-denaturing gels 40
3.4.8 Western blots 41

3.5. Native chemical ligation 41
3.5.1 Purification of semisynthetic STAT6 42
3.5.2 Separation of peptide from ligation product 42

3.6. Bioactivity methods 43
3.6.1 Electrophoretic mobility shift assay 43
3.6.2 Cell culture 44
3.6.3 Microinjection 45
3.6.4 Photo activation 46
3.6.5 Fluorescence measurement 46


Results

4.1 Peptide synthesis 47
4.1.1. Synthesis of Peptide 1 51
4.1.2. Synthesis of Peptide 2 54
4.1.3. Synthesis of Peptide 3 55
4.1.4. Synthesis of Peptide 4 57
4.1.5. Synthesis of Peptide 5 58
4.1.6. Synthesis of Peptide 6 59
4.1.7. Synthesis of Peptide 7 60

4.2 Expressed protein ligation 62
4.2.1 Expression and purification of STAT6MxeCBD protein 62
4.2.2 Preparation of STAT6-1 67
4.2.3 Preparation of STAT6-2 72
4.2.4 Preparation of STAT6-3 73
4.2.5 Preparation of STAT6-4 75
4.2.6 Preparation of STAT6-5 76
4.2.7 Preparation of STAT6-6 77

4.3 Bioactivity of semisynthetic STAT6 80
4.3.1 Decaging of caged phospho tyrosine in STAT6 variants 80
4.3.2 Binding of STAT6 variants to ds DNA 83
4.3.3 Monitoring the STAT6 and ds GAS-DNA by FRET 95
4.3.4 In vivo analysis of STAT6 variants 95

Discussion

5.1 Biological background of STAT6 106
5.2 Caged phosphorylated tyrosine building block 109
5.3 Generation of semisynthetic STAT6 variants 112
5.4 Choice of fluorophore 115
5.5 Bioactivity of STAT6 proteins 116
Summary and Outlook

6.1 Summary 123
6.2 Outlook 125

Acknowledgement 126

Reference list 127

Appendix 144 Abbreviations

aa Amino acids
APS Ammonium persulphate
BOC tert-butyloxycarbonyl
CBD Chitin binding domain
CDI Carbonyl diimidazole
COSR C-terminal thioester
C-terminus Carboxy terminus
DCM Dicholoromethane
dd H O Distilled water 2
DDM n-dodecyl β-D-maltoside
DIEA diisopropyl ethylamine
DMEM Dulbeccos's modified Eagle's medium
DMF Dimethylformamide
DMSO Dimethylsulfoxide
DNA Dioxy ribonucleclic acid
E.coli Escherichia coli
EDTA Ethylene diamine tetraacetate
em Emission
ex Excitation
EPL Expressed protein ligation
ESI-MS Electrospray ionization masspectrometry
FCS Fetal calf serum
Fmoc Fluorenylmethoxycarbonyl
FPLC Fast protein liquid chromatography
FRET Fluorescence resonance energy transfer
HBTU 2-(1H-benzotriazole-1yl)-1,1,3,3
tetramethyluronium hexafluorophosphate
His-tag Histidine tag
HOBT N-hydroxybenzotriazole
HPLC High preformance liquid chromatography Abbreviations

IPTG Isopropyl-β-D-thiogalactopyranoside
JAK Janus kinase
kDa Kilo dalton
MALDI Mattrix assisted laser desorption ionization
MesNa Sodium ß-mercaptoethane sulphonate
MW Molecular weight
NCL Native chemical ligation
NTA Nitrilo tri acetic acid
NPE 1-(2-nitrophenyl) ethanol
N-terminus Amino terminus
OD Optical density
PBS Phosphate buffer saline
rpm Revolution per minute
RT Room temperature
SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel
electrophoresis
SPPS Solid phase peptide synthesis
STAT6 Signal transducer and activator of transcription 6
Tamra Tetra-methyl 6-carboxy rhodamine
TBE Tris Borate buffer
TCEP Tris-(2-carboxyethyl) phosphine
TEMED N, N, N, N-tetramethylethylenediamine
TFA Trifluoracetic acid
TLC Thin layer chromatography
Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol
hydrochloride
UV Ultraviolet
ε Extinction coefficient
λ Wavelength