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

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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.

Sujets

• Biologie

Informations

• Publié par: technische_universitat_carolo-wilhelmina_zu_braunschweig
• Publié le: 01 janvier 2006
• Nombre de lectures: 10
• Langue: English
• Poids de l'ouvrage: 14 Mo

Extrait

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 14 16 17 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 1.1.5 1.1.5.1

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 5 6 6 7 10 10 11 11 12

1

I IV V V

Table of content

1.1.5.3

Abbreviations DNA 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 concept Accessible 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 repertoires Soluble peptide libraries as ligand repertoires

Random mutagenesis Repertoires 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 thesis Material 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 Methods Growth 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 systems Construction of lambda phages presenting model protein domains Comparison of T7 phage with lambda phage Accessible surface scanner software tool Predictions 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 arrays Target-Assisted Iterative Screening (TAIS)

DNA microarray biochip based screening The 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 selection Conclusions and future prospects Literature Appendix Appendix 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  156 163 166 181  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. F 2 Pmp

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