Development of a new platform technology for the recognition and validation of peptide-protein interactions [Elektronische Ressource] / von Andrzej Swistowski
215 Pages
English
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Development of a new platform technology for the recognition and validation of peptide-protein interactions [Elektronische Ressource] / von Andrzej Swistowski

Downloading requires you to have access to the YouScribe library
Learn all about the services we offer
215 Pages
English

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Development of a new platform technology for the recognition and validation of peptide-protein interactions Von der Fakultät für Lebenswissenschaften der Technischen Universität Carolo-Wilhelmina zu Braunschweig zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr.rer.nat.) genehmigte D i s s e r t a t i o n von Andrzej Swistowski aus Klodzko / Polen 1. Referentin oder Referent: Prof. Dr. Jürgen Wehland 2. Referentin oder Referent: Prof. Dr. Stefan Dübel eingereicht am: 13.02.2006 mündliche Prüfung (Disputation) am: 14.06. 2006 2006 (Druckjahr) ́Vorveröffenlichungen der Dissertation Teilergebnisse aus dieser Arbeit wurden mit Genehmigung des Fachbereichs für Biowissenschaften und Psychologie, vertreten durch den Mentor /Betreuer der Arbeit, in folgenden Beiträgen vorab veröffentlicht: Tagungsbeiträge Swistowski A, Bialek K, Beutling U, Frank R. Development of a new biochip platform technology for the recognition and validation of peptide-protein-interactions. 2nd BMBF-Kolloquium „New efficient methods for functional proteome analysis”, 30. May - 1. June 2005, Potsdam (Poster & Abstract) Beutling U, Bialek K, Gupte V, Swistowski A, Rübenhagen R, Frank R. A New Planar Support for Multiple Affinity Enrichment. DECHEMA Statusseminar Chip-Technologien, 3.-4.

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Published 01 January 2006
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Development of a new platform technology for the recognition and validation of peptide-protein interactions Von der Fakultät für Lebenswissenschaften der Technischen Universität Carolo-Wilhelmina zu Braunschweig
von Andrzej Swistowski aus Klodzko / Polen
zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr.rer.nat.) genehmigte D i s s e r t a t i o n
1. Referentin oder Referent: Prof. Dr. Jürgen Wehland 2. Referentin oder Referent: Prof. Dr. Stefan Dübel eingereicht am: 13.02.2006 mündliche Prüfung (Disputation) am: 14.06. 2006 2006 (Druckjahr)
Vorveröffenlichungen der Dissertation Teilergebnisse aus dieser Arbeit wurden mit Genehmigung des Fachbereichs für Biowissenschaften und Psychologie, vertreten durch den Mentor /Betreuer der Arbeit, in folgenden Beiträgen vorab veröffentlicht: Tagungsbeiträge Swistowski A, Bialek K, Beutling U, Frank R.Development of a new biochip platform technology for the recognition and validation of peptide-protein-interactions. 2nd BMBF-Kolloquium „New efficient methods for functional proteome analysis”, 30. May - 1. June 2005, Potsdam (Poster & Abstract) Beutling U, Bialek K, Gupte V, Swistowski A, Rübenhagen R, Frank R. A New Planar Support for Multiple Affinity Enrichment. DECHEMA Statusseminar Chip-Technologien, 3.-4. February, Frankfurt, (Poster & Abstract)
Bialek K, Swistowski A, Mühle L, Frank R. Development of a new biochip platform technology for the recognition and validation of peptide-protein-interactions. 3rd International and 28th European Peptide Symposium. Prague, Czech Republic, September 5-10, 2004. (poster and abstract).
Publikationen/Veröffentlichungen
Bialek K, Swistowski A, Beutling U, Mühle L, Frank R (2004) Development of a New Biochip Platform Technology for the Automated Recognition and Validation of Peptide-Protein-Interactions. In: Solid Phase Synthesis & Combinatorial Libraries 2004́, (Epton, R., ed.) Mayflower Worldwide Ltd., Kingswinford, UK, Chp. 10, 35-38. Bialek K, Swistowski A, Frank R. (2003) Epitope-targeted proteome analysis: towards a
large-scale automated protein-protein-interaction mapping utilizing synthetic peptide arrays. Anal Bioanal Chem., Aug, 376, 7:1006-13, Epub 2003 Apr 03. Frank, R.; Bialek, K., and Swistowski, A. Large-scale protein-interaction mapping with synthetic peptide arrays: epitope-targeted proteome analysis. Peptides 2002; Proceedings 27th European Peptide Symposium, pp. 102-103.
Moim Rodzicom Mojejżonie Ani
Peptide arrays as ligand repertoires
Phage expression libraries of modules
Table of Content
1.
Module repertoires used with the yeast two-hybrid system
1.1.2.7 1.1.3 1.1.3.1
Introduction
1.1.2.2
Phage expression libraries of ligands
13 141617 18 19 21 21 22 22 23 24 24 25 27
Bio-Panning with phage displayed protein ligands and domains
1.1.5.2
1.1.4.2 1.1.51.1.5.1
1.1.5.5
1.1.5.7
Genome/proteome scanning
Peptide and protein repertoires for global analysis of modules
1.1.5.6
1.1.5.4
Sub-library pools for iterative a priori deconvolution
1.1.5.8
2 3 4 566710 10 11 1112
1
I IV V V
Table of content
1.1.5.3
AbbreviationsDNA codes Amino Acid Abbreviations Symbols representing consensus sequences of peptide ligands and protein domains
1.2 1.2.1 1.2.2
Peptide arrays as domain repertoires The experimental conceptAccessible Surface Scanner The SPOT synthesis technique
Repertoires from cell extracts Repertoires of proteins based on expression cloning of DNA libraries Ligand repertoires used with the yeast two-hybrid system
Dedicated peptide libraries Synthetic peptide repertoiresSoluble peptide libraries as ligand repertoires
Random mutagenesisRepertoires of peptide ligands based on expression cloning of oligonucleotide libraries Random peptide libraries
1
1.1.2.4
1.1.2.5
1.1.2.3
Protein arrays as domain repertoires Mutagenized domain libraries Site-directed mutagenesis
Bead-bound peptide libraries as ligand repertoires
1.1.3.2 1.1.4 1.1.4.1
1.1
Protein Scanning Repertoires (Peptide walking)
1.1.1 1.1.2 1.1.2.1
Protein arrays as ligand repertoires
1.1.2.6
Replacement repertoires
Determination of the T7 and lambda bacteriophage titers
2.2.4.3
Affinity selection of phages on peptide synthesized on a plastic foil surface
T7 and lambda phage display systems Aim of this thesisMaterial and methods
33
Isolation of PCR products and plasmid DNA
40 41 42 44 45 45 45 46 46 46 47 47 48 48 49 49 50 50 51 53 53 53 53 54 55
Ligation
DNA digestion
33 33 35 36 37 38 39 40
2.1.5 2.1.6 2.1.7
29 31
2.2.3.8
2.2.3.6
2.2.3.5
2.2.3.7
2.2.3.9
DNA agarose electrophoresis
Dephosphorylation
Table of content
Isolation of phage DNA
Determination of DNA concentration
DNA extraction from agarose gels
2.2.3.4
2.2.3.10 Transformation of E.coli cells
Affinity selection of phages on the SPOT membrane
Preparation of T7select10-3 phage lysates
2.2.4.2
2.2.3.11 Polymerase chain reaction 2.2.4 Phage work 2.2.4.1 Preparation ofλDisplay1 phage lysates
Chemicals/reagents, materials, kits and enzymes
2.2.3.2
2.2.3.3
2.2.1 2.2.2 2.2.3 2.2.3.1
Material
Devices Computer software Bacteria, bacteriophages and clone libraries Vectors Oligonucleotides DNA-Molecular Weight Markers Antibiotics Media for growth of bacteria Buffers and solutions Fmoc-amino acid stock solutions MethodsGrowth of bacteria Storage of bacteria and phages Molecular biology methods Precipitation of DNA
2.1.8 2.1.9 2.1.10 2.1.11 2.2
1.2.3 1.3 2.
2.2.4.5
2.2.4.4
2.1 2.1.1 2.1.2 2.1.3 2.1.4
2.2.4.6 2.2.5 2.2.5.1 2.2.5.2 2.2.5.3 2.2.5.4 2.2.5.5 2.2.5.6 2.2.5.7 2.2.5.8 2.2.5.9
Target Assisted Iterative Screening (TAIS) Fabrication and handling of DNA microarray biochips Preparation of DNA samples for printing Printing of high density DNA microarray Fixation of the DNA features to the slide surface Washing and blocking the slides with BSA solution Chemical blocking with succinic anhydride Cleaning of cover-slips Array quality check hybridization Target to probe hybridization Post hybridization slide processing 2.2.5.10 Biochips scanning Peptide SPOT synthesis on cellulose membranes Protein - SPOT peptide binding assay
2.2.6 2.2.7 2.2.8 3.
3.1 3.1.1 3.1.2 3.2 3.2.1 3.2.2 3.2.3 3.2.4 3.2.5 3.3
3.4 3.5 3.5.1 3.5.2 3.5.3 3.5.4 3.5.5
3.5.6 3.5.7
Expression and purification of high-activity Taq DNA polymerase
Results and discussion
Table of content
Lambda and T7 phage display systemsConstruction of lambda phages presenting model protein domains Comparison of T7 phage with lambda phage Accessible surface scanner software toolPredictions of surface probability for model ligands Predictions of surface probability for different domain-ligands ASS predictions versus protein structure Comparison of the surface probability and solvent accessibility algorithms
Summary High-throughput affinity selection tests of the phage displayed  protein library on the SPOT peptide arraysTarget-Assisted Iterative Screening (TAIS)
DNA microarray biochip based screeningThe concept Proof of principle Choice of the full length cDNA probe library Comparison of plasmid and PCR probes spotted on a glass surface Large scale preparation and spotting of PCR products Functional analysis of microarray experiments Cross-reactivity of the EVH1 domain
55 56 57 57 57
58 58 58 59 59 60 60 60 64 65
66
66 66 70 78 78 85 89 92 96
98
106
113 113 116 120 120 123 131 139
andphage affinity selectionConclusions and future prospectsLiteratureAppendixAppendix 1 Appendix 2 Appendix 3 Appendix 4 Appendix 5 Acknowledgements
Statistical evaluation of microarray results Phage library expansion by infection and propagation in bacteria Expanding the interaction screen to other domains and peptides Conclusions
3.5.8 3.5.9 3.5.10 3.5.11 3.6 4.5.6.
 143  147  149  155 A novel structurally modified plastic surface for peptide synthesis  156163166 181  181  184  185  193  195
Table of content
Abbreviations
Abbreviation A Ab Abl Abp1 Acm AFAP Amp AP APEG R Ap ARVCF BCIP BLAST Boc bp BPB BRCT
BSA C C3G c-ABL CBS CD2 CD2BP2 CDC 42 GAP CDC42 cDNA cfu Cm R Cm COLT CORT Crk Cy3 Cy5 DCE DCM DIC DMF DMSO DNA
Name Adenosine/Desoxyadenosine (-phosphate) Antibody Tyrosine-protein kinase ABL Actin binding protein 1
Acetamidomethyl protecting group Actin filament associated protein Ampicillin Alkaline Phosphatase amino PEG (Poly-ethylene-glycol) spacer Ampicillin resistance
Abbreviations
Armadillo repeat protein deleted in velo-cardio-facial syndrome 5-bromo,4-chloro,3-indolylphosphate Basic Local Alignment Search Tool t-Butyloxycarbonyl protecting group Basepair Bromophenol Blue Conserved domain found at C-terminus of the breast cancer susceptibility gene
Bovine Serum Albumin
Cytidine/desoxycytidine (-phosphate) Guanine nucleotide-releasing factor 2 Abelson murine leukemia viral oncogene homolog 1 Citrate-Buffered Saline T-cell surface antigen CD2 precursor CD2 Cytoplasmic Domain binding protein Cdc42 GTPase-activating protein Cell Division Control protein 42 Complementary DNA Colony Forming Unit Chloramphenicol Chloramphenicol resistant
Cloning of Ligand Targets Cloning of Receptor Targets Proto-oncogene C-crk (P38) Cyanine 3-dCTP Cyanine 5-dCTP 1,2-Dichloroethene Dichloromethane N,N'-Diisopropylcarbodiimide N,N- dimethylformamide
Dimethylsulfoxide
DeoxyriboNucleic Acid
I
Abbreviation
dNTP ds DTT
Dvl1 E. coli EDTA EH ELISA
ErbB-4 EVH1 (E) Ezr f.c. F2Pmp
FACS Fas FE65 (F) FF FHA Fmoc Fyn G GBF GST
GYF h HAc HeLa cells hnRNP K HPLC Hrb HRP HUGE
IL-4 IPTG kb KD KDa KH R Km L. monocytogenes
L.M.P agarose LB
Name Deoxyribonucleoside Triphosphate
Double stranded
Dithiothreitol
Segment polarity protein dishevelled homolog DVL-1
Escherichia coli
Ethylendiamintetraacetate
Eps15 homology domain
Enzyme Linked ImmunoSorbent Assay
Tyrosine kinase erbB-4
Ena-VASP Homology domain 1 Ezrin Final concentration (phosphonodifluoromethyl)-phenylalanine
Fluorescence Activated Cell Sorting Apoptosis-mediating surface antigen FAS Amyloid beta A4 precursor protein-binding family B member 1 A domain with two conserved phenylalanines (F); Forkhead-associated; Fluorenylmethyloxycarbonyl Proto-oncogene tyrosine-protein kinase Fyn
Guanosine/Desoxyguanosine (-phosphate)
Gesellschaft für Biotechnologische Forschung
Glutathione S-transferase
Glycine-tyrosine-phenylalanine protein domain Hour Acetic Acid Cell line taken from Henrietta Lacks alias Helen Lane
Abbreviations
Heterogeneous nuclear ribonucleoprotein F High Performance Liquid Chromatography HIV-1 Rev-binding protein Horse Radish Peroxidase A Database of Human Unidentified Gene-Encoded Large Proteins analyzed by Kazusa human cDNA project Interleukin-4
Isopropyl-β-D-thiogalactopyranoside Kilobase Dissociation constant Kilodalton Ribonucleoprotein K homology Kanamycin resistance Listeria monocytogenes Low Melting Point agarose
Luria-Bertina medium
II
Abbreviation LB-EBV Lin1p MALDI-MS Mena min MTT NMP NUMB OD OPAL OPD OtBu p.a. Cas p130
PBS PCR PDZ PEG
pfu PH PI3K p85 Pmc PMSF POD PPII Prp-8 PTB rpm
RRT RSV RZPD SDS
sec SETA SF 1 SH 1, 2, 3 Sh3yl1 Shb SmB SOS-1 SPR SSC STRAP
Name B Lymphocyte cells immortalized by the Epstein-Barr virus;
Protein LIN1
Matrix Assisted Laser Desorption Ionization Mass Spectrometry Enabled protein homolog Minute Methylthiazolyldiphenyl-tetrazolium-bromide 1-Methyl-2-pyrrolidinone Protein numb homolog Optical density
Oriented Peptide Array Library
o-Phenylene diamine
t-butyl ester Per analysis CRK-associated substrate Phosphate Buffer Saline Polymerase Chain Reaction Protein domain also called Discs-large homologous regions (DHR) Polyethylene Glycol 6000 Plaque Forming Unit Pleckstrin homology PI3-kinase p85-alpha subunit
Abbreviations
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl protecting group phenylmethanesulfonyl-fluoride Peroxidase Polyproline Type II helix
Pre-mRNA splicing factor PRP8 homolog
Phosphotyrosine-binding
Rounds per minute
Recombinant RT-loop
Rous sarcoma virus German Resource Center for Genome Research Sodium Dodecyl Sulfate Second Src homology 3 expressed in tumorigenic astrocytes; Steroidogenic Factor 1 Src homology 1, 2, 3
Sh3 domain YSC-like 1 Ubiquitously expressed Src homology 2 protein snRNP-associated protein Son of sevenless protein homolog 1
Surface Plasmon Resonance Sodium chloride-Sodium Citrate buffer Streptavidin Alkaline Phosphatase complex
III