Expression, purification and biophysical characterization of human Presenilin 2:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
Jülich
Forschungszentrum Jülich, Zentralbibliothek
2013
|
| Schriftenreihe: | Schriften des Forschungszentrums Jülich
Reihe Gesundheit ; 67 |
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | 158 S. Ill., graph. Darst. 24 cm |
| ISBN: | 9783893369287 |
Internformat
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| 245 | 1 | 0 | |a Expression, purification and biophysical characterization of human Presenilin 2 |c Ge Yang |
| 264 | 1 | |a Jülich |b Forschungszentrum Jülich, Zentralbibliothek |c 2013 | |
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Datensatz im Suchindex
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| adam_text | TABLE OF CONTENTS
TABLE OF CONTENTS 3
LIST OF FIGURES 7
LIST OF TABLES 10
1 INTRODUCTION 11
1. L HISTOPATHOLOGY OF ALZHEIMER S DISEASE 11
L .2 GENETICS OF ALZHEIMER S DISEASE 12
1.3 BIOCHEMISTRY OF ALZHEIMER S DISEASEA 13
L .4 COMPONENT OF GAMMA
SECRETASE 14
1.5 STRUCTURE ANALYSIS OF PRESENILIS 16
1.6 OBJECTIVE OF THIS STUDY 18
2 MATERIALS AND METHODS 19
2.1 MATERIALS AND THEIR SOURCE 19
2.1.1 KITS 19
2.1.2 MOLECULAR BIOLOGY MATERIALS 19
2.1.3 RESINS AND COLUMNS 20
2.1.4 INSTRUMENTS 20
2.1.5 BACTERIAL STRAINS, VECTORS ANDPLASMIDS 21
2.1.6 MISCELLANEOUS MATERIALS 22
2.1.7 MEDIA 22
2.1.8 BUFFERS AND SOLUTIONS 23
2.2 METHODS 26
2.2.1 CLONING 26
2.2.1.1 PRINCIPLE 26
2.2.1.2 EXPERIMENT 27
2.2.2 EXPRESSION 29
2.2.3 CELL LYSIS AND FRACTIONATION 29
2.2.4 PURIFICATION 30
2.2.4.1 IMMOBILIZED METAL AFFINITY CHROMATOGRAPHY (IMAC) 30
2.2.4.2 REMOVAL OF PERIPLASMIC FRACTION BY OSMOTIC SHOCK 32
2.2.4.3 STREP-T ACTIN PURIFICATION 33
2.2.4.4 ION EXCHANGE CHROMATOGRAPHY (I EX) 34
2.2.5 AMMONIUM SULPHATE PRECIPITATION (ASP) 35
2.2.5.1 PRINCIPLE 35
2.2.5.2 EXPERIMENT 36
2.2.6 SIZE EXCLUSION CHROMATOGRAPHY (SEC) 37
2.2.6.1 PRINCIPLE OF SEC 37
2.2.6.2 COLUMN CALIBRATION 37
2.2.6.3 SEC MEASUREMENTS 40
2.2.7 MASS SPECTROMETRY (MS) 41
2.2.7.1 PRINCIPLE OF MS 41
2.2.7.2 EXPERIMENT 43
2.2.8 REFOLDING OF HIS-PS2-FL 44
3
HTTP://D-NB.INFO/1058332643
2.2.8.1 PRINCIPLE 44
2.2.8.2 EXPERIMENT 45
2.2.9 PROTEIN CONCENTRATION DETERMINATION 46
2.2.9.1 ULTRAVIOLET/VISIBLE (UV/VIS) SPECTROSCOPY 46
2.2.9.2 THE BRADFORD ASSAY 47
2.2.9.3 EXPERIMENT 47
2.2.10 GEL ELECTROPHORESIS 48
2.2.10.1 PRINCIPLE 48
2.2.10.2 EXPERIMENT 49
2.2.11 WESTERN BLOT 50
2.2.11.1 PRINCIPLE SO
2.2.11.2 EXPERIMENT 50
2.2.12 FLUORESCENCE SPECTROSCOPY 51
2.2.12.1 PRINCIPLE 51
2.2.12.2 EXPERIMENT 53
2.2.13 CIRCULAR DICHROISM (CD) SPECTROSCOPY 54
2.2.13.1 PRINCIPLE OF CD 54
2.2.13.2 CD MEASUREMENTS 57
2.2.14 DYNAMIC LIGHT SCATTERING. 59
2.2.14.1 PRINCIPLE 59
2.2.14.2 MEASUREMENTS 60
2.2.15 RECONSTITUTION AND CRYSTALLIZATION IN MONOOLEIN (MO) CUBIC PHASE
60
2.2.15.1 PRINCIPLE 60
2.2.15.2 EXPERIMENT 61
2.2.16 IN VITRO TRANSLATION 62
2.2.16.1 PRINCIPLE 62
2.2.16.2 EXPERIMENT 63
3 RESULTS 65
3.1 EXPRESSION PURIFICATION AND BIOPHYSICAL CHARACTERIZATION OF
HIS-PS2-FL 65
3.1.1 EXPRESSION 65
3.1.2 PURIFICATION UNDER DENATURING CONDITION 66
3.1.2.1 NI-NTA PURIFICATION 66
3.1.2.2 SIZE EXCLUSION CHROMATOGRAPHY 67
3.1.2.3 REFOLDING OF HIS-PS2-FL 68
3.1.3 PURIFICATION UNDER NATIVE CONDITION 69
3.1.3.1 COMPARISON OF CO-NTA AND NI-NTA 69
3.1.3.2 PURIFICATION WITH DIFFERENT AMOUNT OF NI-NTA 69
3.1.3.3 AMMONIUM SULPHATE PRECIPITATION 70
3.1.3.4 OPTIMIZATION OF PURIFICATION 71
3.1.3.5 ANION EXCHANGE CHROMATOGRAPHY 72
3.1.3.6 SIZE EXCLUSION CHROMATOGRAPHY 74
3.1.4 BIOPHYSICAL CHARACTERIZATION 74
3.1.4.1 MASS SPECTROMETRY 75
3.1.4.2 CIRCULAR DICHROISM SPECTROSCOPY 76
3.1.4.3 FLUORESCENCE SPECTROSCOPY 77
3.1.4.4 RECONSTITUTION INTO MO CUBIC PHASE 77
3.1.4.5 SIZE EXCLUSION CHROMATOGRAPHY 78
3.2 CLONING, EXPRESSION, PURIFICATION AND CHARACTERIZATION OF
PS2-PROTEINS
79
3.2.1 CLONING AND EXPRESSION 79
3.2.2 SOLUBILISATION 80
3.2.2.1 SOLUBILISATION WITH DIFFERENT DETERGENTS 80
3.2.2.2 DOUBLE EXTRACTION WITH SAME DETERGENT 83
3.2.2.3 DOUBLE EXTRACTION WITH DIFFERENT DETERGENTS 84
3.2.2.4 SOLUBILISATION WITH DIFFERENT AMOUNTS OFFC12 MICELLES 85
4
3.2.2.5 SOLUBILISATION WITH DIFFERENT TIME DURATIONS 85
3.2.3 PURIFICATION 86
3.2.3.1 NI-NTA PURIFICATION WITH DIFFERENT DETERGENTS 86
3.2.3.2 STREP-TACTIN PURIFICATION WITH DIFFERENT DETERGENTS 87
3.2.3.3 STREP-TACTIN PURIFICATION WITH DIFFERENT FC12 CONCENTRATIONS 88
3.2.3.4 IMIDAZOLE GRADIENT ELUTION 89
3.2.3.5 NI-NTA PURIFICATION 90
3.2.3.6 STREP-TACTIN PURIFICATION AFTER NI-NTA 90
3.2.3.7 REMOVAL OF PERIPLASMIC FRACTION BY OSMOTIC SHOCK 91
3.2.3.8 NI-NTA PURIFICATION AFTER OSMOTIC SHOCK 92
3.2.3.9 MASS SPECTROMETRY 93
3.2.3.10 SIZE EXCLUSION CHROMATOGRAPHY 94
3.2.3.11 UPSCALED PURIFICATION OF HIS-PS2-NTF-STREP 96
3.2.4 BIOPHYSICAL CHARACTERIZATION OF HIS-PS2-NTF-STREP 98
3.2.4.1 SIZE EXCLUSION CHROMATOGRAPHY 98
3.2.4.2 CD SPECTROSCOPY ANALYSIS OF HIS-PS2-NTF-STREP 101
3.2.4.3 FLUORESCENCE SPECTROSCOPY 108
3.2.4.4 DYNAMIC LIGHT SCATTERING 110
3.2.5 CRYSTALLIZATION OFHIS-PS2-NTF-STREP IN MO LIPIDIC CUBIC PHASE ILL
3.3 IN VITRO TRANSLATION 112
3.3.1 IN VITRO TRANSLATION OF PS2-CONSTRUCTS 112
3.3.2 IN VITRO TRANSLATION OF HIS-PS2-FL-STREP WITH DETERGENTS 112
3.3.3 IN VITRO TRANSLATION OF HIS-PS2-FL-STREP WITH DIFFERENT DETERGENT
CONCENTRATIONS 113
3.3.4 IN VITRO TRANSLATION OF HIS-PS2-FL-STREP WITH PEPTIDES 114
3.3.5 IN VITRO TRANSLATION OF HIS-PS2-FL-STREP WITH PEPTIDES UNDER
OXIDIZING CONDITION 115
3.3.6 IN VITRO TRANSLATION OF PS2-CONSTRUCTS WITH NANODISCS 116
3.3.7 HOMOGENEITY OF IN VITRO TRANSLATED HIS-PS2-CTF-STREP 117
3.3.8 HOMOGENEITY OF IN VITRO TRANSLATED HIS-PS2-CTF-STREP WITH
DETERGENTS 117
3.3.9 HOMOGENEITY OF IN VITRO TRANSLATED HIS-PS2-CTF-STREP WITH
NANODISCS 119
3.3.10 EFFECT OF INCREASED NANODISCS CONCENTRATION ON THE
OLIGOMERISATION OF IN VITRO TRANSLATED
HIS-PS2-CTF-STREP 121
4 DISCUSSION 122
4. L EXPRESSION PURIFICATION AND REFOLDING OF HIS-PS2-FL 122
4.1.1 EXPRESSION 122
4.1.2 PURIFICATION 123
4.1.3 REFOLDING. 124
4.2 SOLUBILISATION, PURIFICATION AND CHARACTERIZATION OF
HIS-PS2-NTF-STREP
124
4.2.1 SOLUBILISATION 124
4.2.2 PURIFICATION 126
4.2.3 BIOPHYSICAL CHARACTERIZATION 127
4.2.3.1 OLIGOMERISATION IN DIFFERENT DETERGENTS 127
4.2.3.2 FAR UV AND NEAR UV CD 128
4.2.3.3 DETERGENTS AND PH DEPENDENCE OF PROTEIN FLUORESCENCE 129
4.2.3.4 FUNCTIONAL IMPLICATION OF DIMERIZATION 130
4.2.3.5 THERMAL STABILITY OF THE DIMER IN FC12 132
4.2.4 CRYSTALLIZATION IN MO CUBIC PHASE 134
4.3 IN VITRO TRANSLATION 134
5 SUMMARY 137
5
ZUSAMMENFASSUNG 139
ABBREVIATIONS 141
BIBLIOGRAPHY 144
APPENDIX I: DNA AND PROTEIN SEQUENCES 152
ACKNOWLEDGEMENTS 155
ERKLARUNG 158
6
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| author | Yang, Ge |
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| dewey-ones | 612 - Human physiology |
| dewey-raw | 612.01575 |
| dewey-search | 612.01575 |
| dewey-sort | 3612.01575 |
| dewey-tens | 610 - Medicine and health |
| discipline | Allgemeine Naturwissenschaft Medizin |
| format | Thesis Book |
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| language | English |
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| physical | 158 S. Ill., graph. Darst. 24 cm |
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| spelling | Yang, Ge Verfasser (DE-588)1045089044 aut Expression, purification and biophysical characterization of human Presenilin 2 Ge Yang Jülich Forschungszentrum Jülich, Zentralbibliothek 2013 158 S. Ill., graph. Darst. 24 cm txt rdacontent n rdamedia nc rdacarrier Schriften des Forschungszentrums Jülich : Reihe Gesundheit 67 Zugl.: Düsseldorf, Univ., Diss., 2012 (DE-588)4113937-9 Hochschulschrift gnd-content Schriften des Forschungszentrums Jülich Reihe Gesundheit ; 67 (DE-604)BV035800060 67 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027534420&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Yang, Ge Expression, purification and biophysical characterization of human Presenilin 2 Schriften des Forschungszentrums Jülich |
| subject_GND | (DE-588)4113937-9 |
| title | Expression, purification and biophysical characterization of human Presenilin 2 |
| title_auth | Expression, purification and biophysical characterization of human Presenilin 2 |
| title_exact_search | Expression, purification and biophysical characterization of human Presenilin 2 |
| title_full | Expression, purification and biophysical characterization of human Presenilin 2 Ge Yang |
| title_fullStr | Expression, purification and biophysical characterization of human Presenilin 2 Ge Yang |
| title_full_unstemmed | Expression, purification and biophysical characterization of human Presenilin 2 Ge Yang |
| title_short | Expression, purification and biophysical characterization of human Presenilin 2 |
| title_sort | expression purification and biophysical characterization of human presenilin 2 |
| topic_facet | Hochschulschrift |
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