Human interleukin-4 binding protein epitope involved in high affinity binding of interleukin-4: molecular analysis and characterisation
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| Main Author: | |
|---|---|
| Format: | Book |
| Language: | English |
| Published: |
2001
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| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Item Description: | Würzburg, Univ., Diss., 2002 |
| Physical Description: | 157 Bl. Ill., graph Darst. : 30 cm |
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MARC
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| 100 | 1 | |a Wietek, Irina |d 1972- |e Verfasser |0 (DE-588)123725569 |4 aut | |
| 245 | 1 | 0 | |a Human interleukin-4 binding protein epitope involved in high affinity binding of interleukin-4 |b molecular analysis and characterisation |c vorgelegt von Irina Wietek |
| 264 | 1 | |c 2001 | |
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Record in the Search Index
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CONTENTS
CONTENTS
1 INTRODUCTION.4
2. MATERIALS AND METHODS.22
2.1 ABBREVIATIONS.22
2.2 CHEMICALS AND ENZYMES.25
2.3 BACTERIAL STRAINS.25
2.4 CELL LINES.25
2.5 ANTIBODIES.26
2.6 VECTORS AND OLIGONUCLEOTIDES.26
2.6.1 EXPRESSION VECTOR FOR E.COLI.26
2.6.2 BACULOVIRUS TRANSFER VECTOR PACGP67-B.26
2.6.3 OLIGONUCLEOTIDES.26
2.7 MICROBIOLOGICAL METHODS.28
2.7.1 STERILIZATION.28
2.7.2 CULTURE MEDIA.29
2.7.3 CULTURING OF BACTERIA.29
2.7.4 ELECTROCOMPETENT E.COLI.30
2.7.4.1 PREPARATION OF ELECTROCOMPETENT BACTERIAL CELLS.30
2.7.4.2 ELECTROTRANSFORMATION OF COMPETENT E.COLI.30
2.7.5 STORAGE OF BACTERIAL CULTURES.30
2.8 MOLECULAR BIOLOGICAL METHODS.31
2.8.1 DETERMINATION OF THE CONCENTRATION OF NUCLEIC ACIDS.31
2.8.2 PHENOL EXTRACTION OF DNA. 31
2.8.3 ETHANOL PRECIPITATION OF DNA.31
2.8.4 DNA MOLECULAR STANDARDS.32
2.8.5 DNA AGAROSE GEL ELECTROPHORESIS.32
2.8.6 PURIFICATION OF DNA BY AGAROSE GEL ELECTROPHORESIS.33
2.8.7 SITE-DIRECTED MUTAGENESIS BY PCR.33
2.8.8 SITE-DIRECTED MUTAGENESIS USING DNA CASSETTES.35
2.8.9 DIGESTION OF DNA.35
2.8.10 LIGATION OF DNA.36
2.8.11 PREPARATION OF PLASMID DNA.36
2.8.11.1 LYSOZYME-ALKALINE-LYSIS PROCEDURE.36
2.8.11.2 PEG PRECIPITATION PROCEDURE.37
2.8.11.3 PREPARATION OF PLASMID DNA WITH LICL.38
2.8.12 DNA SEQUENCING.39
2.9 PROTEIN CHEMICAL METHODS.39
2.9.1 DETERMINATION OF THE PROTEIN CONCENTRATION.39
2.9.2 LYOPHILIZATION OF PROTEINS.40
2.9.3 MOLECULAR WEIGHT STANDARD FOR PROTEIN SAMPLES.40
1
BIBLIOGRAFISCHE INFORMATIONEN
HTTP://D-NB.INFO/964235471
_CONTENTS
2.9.4 SDS - POLYACRYLAMIDE GEL ELECTROPHORESIS.40
2.9.5 CONCENTRATION OF PROTEIN SAMPLES BY TCA.41
2.9.6 BIOTINYLATION OF PROTEINS.42
2.10 IMMUNOLOGICAL METHODS - WESTERN BLOT.42
2.10.1 TRANSFER OF PROTEINS TO A NITROCELLULOSE MEMBRANE.42
2.10.2 DETECTION OF WESTERN BLOTS BY A PEROXIDASE COUPLED ANTIBODY.43
2.11 EXPRESSION OF RECOMBINANT PROTEINS IN E.COLI.44
2.11.1 TEMPERATURE INDUCED PROTEIN EXPRESSION.44
2.11.2 PREPARATION OF INCLUSION BODIES.45
2.11.3 DENATURATION AND RENATURATION OF PROTEINS.45
2.12 EXPRESSION OF RECOMBINANT PROTEINS IN SF9 CELLS.46
2.12.1 GENERAL HANDLING TECHNIQUES.46
2.12.1.1 INSECT CELL CULTURE MEDIA.46
2.12.1.2 CULTIVATION OF SF9 CELLS.46
2.12.1.3 STAINING AND COUNTING OF SF9 CELLS.47
2.12.1.4 LONG-TERM CELL STORAGE.47
2.12.1.5 MONOLAYER CULTURE.48
2.12.1.6 SUSPENSION CULTURE.48
2.12.2 GENERATION OF RECOMBINANT BACULOVIRUSES.49
2.12.2.1 THE BACULOVIRUS EXPRESSION SYSTEM.49
2.12.2.2 CO-TRANSFECTION OF BACULOGOLD DNA AND A TRANSFER VECTOR INTO
INSECT CELLS.49
2.12.2.3 GENERATING PURE VIRUS STOCKS BY PLAQUE PURIFICATION.51
2.12.3 AMPLIFICATION OF VIRUS STOCKS.52
2.12.3.1 VIRUS AMPLIFICATION FROM A SINGLE VIRUS PLAQUE.52
2.12.3.2 VIRUS AMPLIFICATION IN A SMALL VOLUME SCALE.53
2.12.3.3 VIRUS AMPLIFICATION IN A LARGE VOLUME SCALE.53
2.12.3.4 DETERMINATION OF VIRUS TITER BY PLAQUE ASSAY. 53
2.12.3.5 VIRUS STORAGE.54
2.12.4 PROTEIN EXPRESSION.54
2.13 PURIFICATION OF RECOMBINANT PROTEINS.55
2.13.1 PURIFICATION OF PROTEINS EXPRESSED IN E.COLI.55
2.13.1.1 PROTEIN PURIFICATION BY ION EXCHANGE CHROMATOGRAPHY.55
2.13.1.2 PROTEIN PURIFICATION BY RP-HPLC.55
2.13.2 PURIFICATION OF PROTEINS EXPRESSED IN SF9 CELLS.57
2.13.2.1 AFFINITY CHROMATOGRAPHY USING IL-4 AS A LIGAND.57
2.13.2.2 AFFINITY CHROMATOGRAPHY USING X14/38 AS A LIGAND.57
2.13.2.3 DIALYSIS.58
2.13.2.4 CONCENTRATION OF PROTEINS BY ULTRAFILTRATION.58
2.14 ANALYSIS OF PROTEIN-PROTEIN INTERACTIONS BY BIACORE TECHNOLOGY.59
2.14.1 IMMOBILIZATION OF PROTEINS BY STREPTAVIDIN-BIOTIN COUPLING.60
2.14.2 MEASURING AND EVALUATION OF PROTEIN-PROTEIN INTERACTIONS.60
2.14.3 ASSESSMENT OF THE ENERGETIC EFFECTS OF INTRODUCED MUTATIONS.61
3. RESULTS.62
3.1 PREPARATION OF RECOMBINANT IL-4BP AND ITS VARIANTS.62
3.1.1 CLONING OF IL-4BP AND ITS VARIANTS IN THE BACULOVIRUS TRANSFER
VECTOR PACGP67B.64
O
CONTENTS
3.1.2 CO-TRANSFECTION INTO SF9 INSECT CELLS AND AMPLIFICATION OF THE
RECOMBINANT VIRUS.68
3.1.3 EXPRESSION AND PURIFICATION OF RECOMBINANT IL-4BP AND ITS
VARIANTS.69
3.1.3.1 PURIFICATION OF IL-4BP AND ITS VARIANTS BY IL-4 AFFINITY
COLUMN.69
3.1.3.2 PURIFICATION OF IL-4BP VARIANTS THROUGH ANTIBODY COLUMN.74
3.1.4 BIOTINYLATION OF IL-4BP RECOMBINANT VARIANTS.77
3.2 PREPARATION OF RECOMBINANT IL-4 VARIANTS.78
3.2.1 CLONING OF IL-4 MUTANT VARIANTS.78
3.2.2 EXPRESSION AND PURIFICATION OF IL-4 VARIANTS.81
3.3 KINETIC ANALYSIS OF THE INTERACTION OF IL-4 WITH THE IL-4BP VARIANTS
USING BIACORE
TECHNOLOGY.84
3.3.1 IMMOBILIZATION OF THE BIOTINYLATED IL-4BP AND ITS VARIANTS ON THE
SENSOR CHIPS.84
3.3.2 KINETICS OF THE INTERACTION OF IL-4 AND IL-4BP.87
3.3.3 EFFECTS OF SITE-SPECIFIC PERTURBATIONS IN IL-4BP ON THE
INTERACTION WITH IL-4.89
3.3.3.1 KINETICS OF THE BINDING OF IL-4 TO THE ALANINE VARIANTS OF
IL-4BP.89
3.3.3.2 THERMODYNAMIC ASPECTS OF THE INTERACTION BETWEEN IL-4 AND THE
IL-4BP VARIANTS
INCLUDED IN THE ALANINE SCANNING MUTAGENESIS.99
3.3.3.3 ANALYSIS OF THE BINDING OF IL-4 TO THE IL-4BP TYROSINE
VARIANTS.103
3.3.4 ANALYSIS OF IL-4BP DOUBLE MUTANTS.105
3.4 BIOSENSOR ANALYSIS OF THE KINETICS OF BINDING OF IL-4 VARIANTS TO
THE IL-4BP.110
4. DISCUSSION.112
4.1 STRUCTURAL FEATURES AND BINDING SITE OF HUMAN IL-4BP.112
4.2 DEFINING FUNCTIONAL IMPORTANT RESIDUES FROM THE INTERFACE OF HUMAN
IL-4BP FOR THE
INTERACTION WITH IL-4.116
4.3 ADDITIVITY AND COOPERATIVITY IN THE FUNCTIONAL EPITOPE OF IL-4BP.124
4.4 COMPARISON OF THE STRUCTURAL AND THE FUNCTIONAL IL-4BP EPITOPES.126
4.5 COMPLEMENTARITY BETWEEN THE FUNCTIONAL EPITOPES OF HUMAN IL-4 AND
IL-4BP.129
4.6 COMPARISON OF THE IL-4BP FUNCTIONAL EPITOPE TO OTHER CYTOKINE
RECEPTORS.131
5. SUMMARY. 135
6. LITERATURE.137
7. APPENDICES.146
ACKNOWLEDGEMENTS.155
LEBENSLAUF.156
ERKLARUNG.157 |
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| indexdate | 2024-10-09T18:05:09Z |
| institution | BVB |
| language | English |
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| physical | 157 Bl. Ill., graph Darst. : 30 cm |
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| spelling | Wietek, Irina 1972- Verfasser (DE-588)123725569 aut Human interleukin-4 binding protein epitope involved in high affinity binding of interleukin-4 molecular analysis and characterisation vorgelegt von Irina Wietek 2001 157 Bl. Ill., graph Darst. : 30 cm txt rdacontent n rdamedia nc rdacarrier Würzburg, Univ., Diss., 2002 Interleukin 4 (DE-588)4212826-2 gnd rswk-swf Mensch (DE-588)4038639-9 gnd rswk-swf Molekularbiologie (DE-588)4039983-7 gnd rswk-swf Bindeproteine (DE-588)4276933-4 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Mensch (DE-588)4038639-9 s Interleukin 4 (DE-588)4212826-2 s Bindeproteine (DE-588)4276933-4 s Molekularbiologie (DE-588)4039983-7 s DE-604 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=009824443&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Wietek, Irina 1972- Human interleukin-4 binding protein epitope involved in high affinity binding of interleukin-4 molecular analysis and characterisation Interleukin 4 (DE-588)4212826-2 gnd Mensch (DE-588)4038639-9 gnd Molekularbiologie (DE-588)4039983-7 gnd Bindeproteine (DE-588)4276933-4 gnd |
| subject_GND | (DE-588)4212826-2 (DE-588)4038639-9 (DE-588)4039983-7 (DE-588)4276933-4 (DE-588)4113937-9 |
| title | Human interleukin-4 binding protein epitope involved in high affinity binding of interleukin-4 molecular analysis and characterisation |
| title_auth | Human interleukin-4 binding protein epitope involved in high affinity binding of interleukin-4 molecular analysis and characterisation |
| title_exact_search | Human interleukin-4 binding protein epitope involved in high affinity binding of interleukin-4 molecular analysis and characterisation |
| title_full | Human interleukin-4 binding protein epitope involved in high affinity binding of interleukin-4 molecular analysis and characterisation vorgelegt von Irina Wietek |
| title_fullStr | Human interleukin-4 binding protein epitope involved in high affinity binding of interleukin-4 molecular analysis and characterisation vorgelegt von Irina Wietek |
| title_full_unstemmed | Human interleukin-4 binding protein epitope involved in high affinity binding of interleukin-4 molecular analysis and characterisation vorgelegt von Irina Wietek |
| title_short | Human interleukin-4 binding protein epitope involved in high affinity binding of interleukin-4 |
| title_sort | human interleukin 4 binding protein epitope involved in high affinity binding of interleukin 4 molecular analysis and characterisation |
| title_sub | molecular analysis and characterisation |
| topic | Interleukin 4 (DE-588)4212826-2 gnd Mensch (DE-588)4038639-9 gnd Molekularbiologie (DE-588)4039983-7 gnd Bindeproteine (DE-588)4276933-4 gnd |
| topic_facet | Interleukin 4 Mensch Molekularbiologie Bindeproteine Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=009824443&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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