Structural and biophysical characterization of antibody:antigen complexes of therapeutic relevance:
Gespeichert in:
| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2013
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| Schlagworte: | |
| Online-Zugang: | Volltext https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20130429-1131433-0-7 Inhaltsverzeichnis |
| Beschreibung: | VI, 161 Bl. Ill., graph. Darst. |
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| 245 | 1 | 0 | |a Structural and biophysical characterization of antibody:antigen complexes of therapeutic relevance |c Felix Ludwig Schiele |
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Datensatz im Suchindex
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| adam_text | IMAGE 1
CONTENTS
CONTENTS
1 INTRODUCTION 1
1.1 ANTIBODIES 1
1.1.1 IMMUNE SYSTEM AND GENERATION OF ANTIBODIES 1
1.1.2 STRUCTURE OF ANTIBODIES 2
1.1.3 ANTIBODIES AND FABS FOR THERAPEUTIC USE 5
1.2 DABIGATRAN ANTIDOTE 9
1.2.1 THE COAGULATION CASCADE 9
1.2.2 ANTICOAGULANTS AND ANTIDOTES 11
1.2.3 AN ANTIDOTE FOR DABIGATRAN 12
1.3 PCSK9 - A TARGET FOR DYSLIPIDEMIA 14
1.3.1 BIOLOGICAL FUNCTION OF PCSK9 14
1.3.2 STRUCTURE OF PCSK9 16
1.3.3 INHIBITION OF PCSK9 FUNCTION 18
2 THESIS MOTIVATION AND SUMMARY 19
3 MATERIAL 21
3.1 CHEMICALS 21
3.2 MEDIA 21
3.3 BUFFERS . 21
3.4 CRYSTALLIZATION MATERIAL 22
3.5 MATERIALS FOR PROTEIN EXPRESSION AND PURIFICATION 23
3.6 SOFTWARE 26
3.7 X-RAY EQUIPMENT 26
4 METHODS 28
4.1 MOLECULAR BIOLOGICAL METHODS 28
4.1.1 PREPARATION OF PLASMID AND LINEARIZED DNA 28
4.1.2 DNA RESTRICTION BY ENDONUCLEASES 28
4.1.3 AGAROSE GEL ELECTROPHORESIS 29
4.1.4 LIGATION 29
4.1.5 TRANSFORMATION AND CULTURE OF E. COLI BACTERIA 30
4.1.6 DNA SEQUENCING 30
4.2 CELL CULTURE METHODS 31
4.2.1 CULTIVATION AND TRANSIENT TRANSFECTION OF HEK293-6E CELLS . . 31
4.2.2 CULTIVATION OF HYBRIDOMAS 32
4.2.3 EXPRESSION OF ADABI-MAB2 IN CHOS 32
HTTP://D-NB.INFO/1038329094
IMAGE 2
CONTENTS
4.3 PROTEIN PREPARATION 33
4.3.1 EXPRESSION AND PURIFICATION OF PCSK9 33
4.3.2 EXPRESSION AND PURIFICATION OF PC SK9ACTD 34
4.3.3 PURIFICATION OF FULL-LENGTH ANTIBODIES 35
4.3.4 PAPAIN CLEAVAGE OF ANTIBODIES - PREPARATION OF FABS 36
4.3.5 SEPARATION OF ANTIBODY FRAGMENTS 37
4.3.6 PURIFICATION OF ANTI-DABIGATRAN ANTIBODY FRAGMENTS 37
4.4 CHARACTERIZATION OF PROTEINS 38
4.4.1 DETERMINATION OF PROTEIN CONCENTRATION BY PHOTOMETRY 38
4.4.2 SDS-PAGE 39
4.4.3 ELISA 40
4.5 PROTEIN CRYSTALLOGRAPHY 41
4.5.1 CRYSTAL LATTICE AND LATTICE SYMMETRY 41
4.5.2 SOLVENT CONTENT OF PROTEIN CRYSTALS 41
4.5.3 CRYSTALLIZATION OF PROTEINS 42
4.5.4 SEEDING METHODS 43
4.5.5 COCRYSTALLIZATION AND SOAKING 44
4.5.6 CRYOBUFFER AND CRYSTAL MOUNTING 45
4.6 X-RAY STRUCTURE SOLUTION . . . 45
4.6.1 THEORY OF X-RAY DIFFRACTION 45
4.6.2 RECIPROCAL LATTICE AND EWALD SPHERE 50
4.6.3 TEMPERATURE FACTORS 51
4.6.4 PATTERSON FUNCTION 53
4.6.5 DATA COLLECTION 54
4.6.6 PROCESSING, SCALING AND REDUCTION OF DATA 55
4.6.7 PHASING BY MOLECULAR REPLACEMENT 56
4.6.8 MODEL BUILDING 58
4.6.9 REFINEMENT 59
4.6.10 STRUCTURE VALIDATION 63
4.6.11 STRUCTURE VISUALIZATION AND ANALYSIS 64
4.7 MOLECULAR DYNAMICS SIMULATION 64
4.8 BIOPHYSICAL METHODS 66
4.8.1 SURFACE PLASMON RESONANCE 66
4.8.2 ISOTHERMAL TITRATION CALORIMETRY . . 70
4.8.3 KINETIC EXCLUSION ASSAY 72
IMAGE 3
CONTENTS
5 RESULTS AND DISCUSSION - DABIGATRAN FABS 75
5.1 ADABI-FABL 75
5.1.1 ANTIBODY SCREENING AND ENGINEERING 75
5.1.2 CRYSTALLIZATION OF FAB 1 76
5.1.3 OVERALL STRUCTURE OF THE COMPLEX 77
5.1.4 CRYSTAL STRUCTURE EXPLAINS HIGH AFFINITY 78
5.1.5 APO STRUCTURE OF ADABI-FABL 80
5.1.6 STRUCTURAL DIFFERENCES BETWEEN THE PROTOMERS 81
5.1.7 CONFORMATIONAL CHANGES BETWEEN APO AND HOLO STRUCTURE ... 82
5.1.8 FAB MIMICS STRUCTURAL FEATURES OF THROMBIN 83
5.2 STRUCTURE ACTIVITY RELATIONSHIP STUDIES 87
5.2.1 SAR STUDIES 90
5.2.2 CORRELATION OF AFFINITY FOR ADABI-FABL AND THROMBIN 93
5.3 ADABI-FAB2 AND 3 94
5.3.1 PURIFICATION OF ADABI-FAB2/3 94
5.3.2 CRYSTALLIZATION OF ADABI-FAB2/3 94
5.3.3 OVERALL STRUCTURE OF THE FABS 95
5.3.4 BINDING MODE OF THE FABS 96
5.3.5 APO STRUCTURES OF THE FABS 98
5.3.6 REDUCED AFFINITY COMPARED TO ADABI-FABL 99
5.3.7 CONFORMATIONAL CHANGES BETWEEN APO AND COMPLEX STRUCTURE . 100
5.3.8 THERMODYNAMICS OF BINDING OF DABIGATRAN TO FABS AND THROM BIN 101
6 RESULTS AND DISCUSSION - PCSK9 104
6.1 PURIFICATION OF PCSK9 104
6.2 APCSK9-FABL 106
6.2.1 EXPRESSION, PURIFICATION AND PAPAIN CLEAVAGE OF APCSK9-MABL 106
6.2.2 EPITOPE IDENTIFICATION BY PEPTIDE MICROARRAYS 106
6.2.3 APCSK9-FABL APO STRUCTURE 107
6.2.4 APCSK9-FABL COMPLEX STRUCTURE 107
6.2.5 CONFORMATIONAL DIFFERENCES BETWEEN APO AND HOLO STRUCTURE . 109
6.2.6 SPR PEPTIDE ANALYSIS 110
6.2.7 APCSK9-MABL DOES NOT BLOCK EGF-A BINDING 113
6.3 APCSK9-FAB2 115
6.3.1 EXPRESSION, PURIFICATION AND PAPAIN CLEAVAGE OF APCSK9-MAB2 115
6.3.2 SPR ANALYSIS 115
6.3.3 APO STRUCTURE OF ADABI-FAB2 116
IMAGE 4
LIST OF TABLES
6.3.4 EXPRESSION AND PURIFICATION OF FAB3H42 117
6.3.5 TERNARY COMPLEX OF ADABI-FAB2 117
6.3.6 MODE OF ACTION OF APCSK9-MAB2 119
6.4 APCSK9-FAB3 122
6.4.1 EXPRESSION, PURIFICATION AND PAPAIN CLEAVAGE OF APCSK9-MAB3 122
6.4.2 CRYSTAL STRUCTURE OF APCSK9-FAB3 122
7 DISCUSSION - FLEXIBILITY OF CDR:H3 125
A APPENDIX 154
LIST OF TABLES
1 COMPOSITION OF ANALYTICAL AND PREPARATIVE DNA DIGESTIONS WITH
ENDONUCLEASES 29
2 COMPOSITION OF TRANSFECTION SOLUTION FOR 100 ML CULTURE VOLUME ... 31
3 PARAMETERS OF SEC ON A HILOAD-26/60-SUPERDEX-200 34
4 PARAMETERS OF SEC ON A HILOAD-26/60-SUPERDEX-75 35
5 INCUBATION TIMES FOR PAPAIN CLEAVAGE 36
6 PARAMETERS OF AFFINITY CHROMATOGRAPHY ON MABSELECT SURE 37
7 MOLAR MASSES AND EXTINCTION COEFFICIENTS OF PROTEINS USED 39
8 CONCENTRATIONS FOR AFFINITY DETERMINATION WITH KINEXA 75
LIST OF FIGURES
1 STRUCTURAL FEATURES OF ANTIBODIES 3
2 FORMATS OF THERAPEUTIC PROTEINS BASED ON ANTIBODIES 8
3 COAGULATION PATHWAYS 10
4 STRUCTURE AND MODE OF ACTION OF DABIGATRAN 12
5 FUNCTION AND STRUCTURE OF PCSK9 15
6 DIFFRACTION OF X-RAYS AND BRAGG S INTERPRETATION 46
7 EWALD SPHERE AND INCIDENCE OF X-RAY REFLECTIONS 51
8 BASIC PRINCIPLES OF SURFACE PLASMON RESONANCE 67
9 BASIC PRINCIPLES OF ISOTHERMAL TITRATION CALORIMETRY 71
10 PRINCIPLE OF KINETIC EXCLUSION ASSAY 73
11 STRUCTURE OF ADABI-FABL 79
12 MOLECULAR DYNAMICS SIMULATION OF ADABI-FABL 83
13 ADABI-FABL MIMICS STRUCTURAL FEATURES OF THROMBIN 84
IMAGE 5
LIST OF FIGURES
14 ADABI-FABL DOES NOT MIMIC FUNCTIONAL FEATURES OF THROMBIN 86 15
ADABI-FABL DOES NOT FUNCTIONALLY MIMIC THROMBIN 86
16 CHEMICAL STRUCTURES OF DABIGATRAN AND STRUCTURAL ANALOGS 88
17 AFFINITY DETERMINATION OF DABIGATRAN ANALOGS FOR THROMBIN BY SPR . .
89 18 AFFINITY BINNING OF DABIGATRAN ANALOGS 90
19 AFFINITY DATA OF DABIGATRAN ANALOGS FOR ADABI-FABL 91
20 CRYSTAL STRUCTURES OF ADABI-FABL IN COMPLEX WITH DABIGATRAN ANALOGS .
92 21 STRUCTURE ACTIVITY RELATIONSHIP OF DABIGATRAN ANALOGS 94
22 PURIFICATION OF ADABIFAB2 AND 3 95
23 CRYSTAL STRUCTURES OF ADABI-FAB2/3 IN COMPLEX WITH DABIGATRAN .... 97
24 CONFORMATIONAL CHANGES BETWEEN APO AND COMPLEX STRUCTURES OF
ADABIFAB2/3 99
25 MOLECULAR DYNAMICS SIMULATION OF CDR-.H3 AFTER REMOVAL OF DABIGA
TRAN FROM THE HOLO STRUCTURE 101
26 THERMODYNAMICS OF DABIGATRAN BINDING 102
27 PURIFICATION OF PCSK9 PROTEINS 105
28 PREPARATION OF FABS FROM APCSK9-MABL 106
29 CRYSTAL STRUCTURES OF APCSK9-FABL 108
30 SPR SENSOGRAMS OF PEPTIDES BINDING TO APCSK9-FABL ILL
31 AFFINITY OF APCSK9-FABL FOR PCSK9 PEPTIDES 112
32 MODELED COMPLEX OF APCSK9-FABL:PCSK9 113
33 BINDING OF PCSK9 AND PCSK9:EGF-A TO MABL 114
34 PREPARATION OF APCSK9-FAB2 BY PAPAIN CLEAVAGE AND STRUCTURAL FEA
TURES OF THE FAB 115
35 MAB2 DOES NOT AFFECT BINDING OF EGF-A PCSK9 116
36 TERNARY COMPLEX OF ADABI-FAB2 118
37 MODE OF ACTION OF APCSK9-FAB2 121
38 PREPARATION OF FABS FROM APCSK9-MAB3 122
39 X-RAY STRUCTURE OF ADABI-FAB3 123
40 CONFORMATIONAL DIFFERENCES OF THE PARATOPE BETWEEN APO AND HOLO
STRUCTURES OF VARIOUS ANTIBODIES 125
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| spelling | Schiele, Felix Verfasser aut Structural and biophysical characterization of antibody:antigen complexes of therapeutic relevance Felix Ludwig Schiele 2013 VI, 161 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier München, Techn. Univ., Diss., 2013 Antikörper (DE-588)4002290-0 gnd rswk-swf Cholesterinstoffwechsel (DE-588)4147860-5 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Antikörper (DE-588)4002290-0 s Cholesterinstoffwechsel (DE-588)4147860-5 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:91-diss-20130429-1131433-0-7 http://mediatum.ub.tum.de/node?id=1131433 Verlag kostenfrei Volltext https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20130429-1131433-0-7 Resolving-System DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026013261&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Schiele, Felix Structural and biophysical characterization of antibody:antigen complexes of therapeutic relevance Antikörper (DE-588)4002290-0 gnd Cholesterinstoffwechsel (DE-588)4147860-5 gnd |
| subject_GND | (DE-588)4002290-0 (DE-588)4147860-5 (DE-588)4113937-9 |
| title | Structural and biophysical characterization of antibody:antigen complexes of therapeutic relevance |
| title_auth | Structural and biophysical characterization of antibody:antigen complexes of therapeutic relevance |
| title_exact_search | Structural and biophysical characterization of antibody:antigen complexes of therapeutic relevance |
| title_full | Structural and biophysical characterization of antibody:antigen complexes of therapeutic relevance Felix Ludwig Schiele |
| title_fullStr | Structural and biophysical characterization of antibody:antigen complexes of therapeutic relevance Felix Ludwig Schiele |
| title_full_unstemmed | Structural and biophysical characterization of antibody:antigen complexes of therapeutic relevance Felix Ludwig Schiele |
| title_short | Structural and biophysical characterization of antibody:antigen complexes of therapeutic relevance |
| title_sort | structural and biophysical characterization of antibody antigen complexes of therapeutic relevance |
| topic | Antikörper (DE-588)4002290-0 gnd Cholesterinstoffwechsel (DE-588)4147860-5 gnd |
| topic_facet | Antikörper Cholesterinstoffwechsel Hochschulschrift |
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