Protein-protein interactions in Arabidopsis thaliana molybdenum cofactor biosynthesis and allocation:
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
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2010
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| Beschreibung: | 88 S. Ill., graph. Darst. |
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Datensatz im Suchindex
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| adam_text | PROTEIN-PROTEIN INTERACTIONS IN ARABIDOPSIS THALIANA MOLYBDENUM COFACTOR
BIOSYNTHESIS AND ALLOCATION VON DER FAKULTAET FUER LEBENSWISSENSCHAFTEN
DER TECHNISCHEN UNIVERSITAET CAROLO-WILHELMINA ZU BRAUNSCHWEIG ZUR
ERLANGUNG DES GRADES EINES DOKTORS DER NATURWISSENSCHAFTEN (DR. RER.
NAT.) GENEHMIGTE DISSERTATION VON MIRCO GEISLER AUS HAMBURG MIRCO
GEISLER TABLE OF CONTENTS TABLE OF CONTENTS 1 INTRODUCTION 1 1.1
MOLYBDENUM AND ITS RELEVANCE IN NATURE 1 1.2 CHEMISTRY OF THE MOLYBDENUM
COFACTOR 2 1.3 SYNTHESIS OF THE MOLYBDENUM COFACTOR 3 1.3.1 STEP 1: THE
FORMATION OF CYCLIC PYRANOPTERIN MONOPHOSPHATE 3 1.3.2 STEP 2: THE
GENERATION OF MOLYBDOPTERIN 4 1.3.3 STEP 3: THE ADENYLATION OF
MOLYBDOPTERIN AND INSERTION OF MOLYBDENUM 4 1.3.4 STEP 4: MODIFICATIONS
OF THE MOLYBDENUM COFACTOR 5 1.4 STORAGE AND INSERTION OF THE MOLYBDENUM
COFACTOR 7 1.5 MOLYBDENUM CONTAINING ENZYMES 7 1.6 PROTEIN-PROTEIN
INTERACTIONS IN MOCO BIOSYNTHESIS 8 1.7 AIM OF THIS WORK 10 2 RESULTS 11
2.1 HETEROLOGOUS EXPRESSION AND PURIFICATION OF RECOMBINANT PROTEINS 11
2.1.1 HETEROLOGOUS EXPRESSION AND PURIFICATION OF HIS-TAGGED PROTEINS IN
ESCHERICHIA COLI 11 2.1.2 HETEROLOGOUS EXPRESSION AND PURIFICATION OF
HIS-TAGGED XDH1, AO1 AND AO3 IN PICHIA PASTORIS 13 2.2 DETECTION OF
PROTEIN-PROTEIN INTERACTIONS BY CHEMICAL CROSSLINKING WITH BIOTIN LABEL
TRANSFER 14 2.2.1 OPTIMIZATION OF CROSSLINKING EXPERIMENTS 15 2.2.2
INTERACTIONS BETWEEN CNX1E AND CNX1G DOMAIN WITH THE LARGE SUBUNIT OF
MPT-SYNTHASE CNX6 16 2.2.2.1 DETERMINATION OF THE CNX1E / CNX6
INTERACTION SITE BY MALDI-TOF MS ANALYSIS 18 2.2.3 PROTEIN INTERACTIONS
BEYOND MOCO BIOSYNTHESIS 19 2.2.3.1 INTERACTION OF ABA3 WITH CNX1 19
2.2.3.2 INTERACTION OF SULFITE OXIDASE WITH CNX1 20 2.2.3.3 INTERACTION
OF SULFITE OXIDASE WITH ABA3-CT 21 MIRCO GEISLER TABLE OF CONTENTS
2.2.3.4 INTERACTION OF CNX1 E AND ABA3-CT WITH XDH 23 2.2.4 INTERACTIONS
OF CNX1E AND ABA3-CT WITH LDP PROTEINS 25 2.3 DETECTION OF IN VIVO
PROTEIN-PROTEIN INTERACTIONS OF MOCO BIOSYNTHETIC PROTEINS 29 2.3.1
CLONING OF PXCSG-STREP 29 2.3.2 EXPRESSION AND PURIFICATION OF PROTEINS
IN NICOTIANA BENTHAMIANA 30 2.3.3 IDENTIFICATION OF CO-PURIFIED PROTEINS
34 2.3.4 IDENTIFICATION OF CO-PURIFIED PROTEINS PLUS IN VIVO
CROSSLINKING 41 2.4 GELFILTRATION ANALYSIS 43 2.5 SURFACE PLASMON
RESONANCE (SPR) SPECTROSCOPY 43 2.6 ISOTHERMAL TITRATION CALORIMETRY
(ITC) 44 3 OISCUSSION 47 3.1 PROTEIN-PROTEIN INTERACTIONS CAN BE SHOWN
BY CHEMICAL CROSSLINKING WITH BIOTIN LABEL TRANSFER 47 3.1.1 INTERACTION
STUDIES OF THE CNX1 DOMAINS CNX1E AND CNX1G WITH THE LARGE SUBUNIT OF
MPT SYNTHASE CNX6 48 3.1.2 INTERACTION STUDIES OF THE CNX1E DOMAIN WITH
ABA3-CT 49 3.1.3 INTERACTION STUDIES OF THE MOCO USER ENZYME XANTHIN
DEHYDROGENASE WITH CNX1 E AND ABA3-CT 50 3.1.4 INTERACTION STUDIES OF
THE MOCO USER ENZYME SULFITE OXIDASE WITH CNX1 E AND ABA3-CT 51 3.1.5
INTERACTION STUDIES OF CNX1 E AND ABA3-CT WITH LDP1 TO LDP8 52 3.2
DETECTION OF PROTEIN-PROTEIN INTERACTIONS IN VIVO 52 3.2.1 PROTEIN
CO-PURIFICATION WITH OVEREXPRESSED A. THALIANA CNX6 IN N. BENTHAMIANA 53
3.2.2 PROTEIN CO-PURIFICATION WITH OVEREXPRESSED A. THALIANA CNX1 IN N.
BENTHAMIANA 54 3.2.3 PROTEIN CO-PURIFICATION WITH OVEREXPRESSED A.
THALIANA CNX1E IN N. BENTHAMIANA 54 3.3 CO-PURIFICATION AND IN VIVO
CROSSLINKING 55 3.4 BIOPHYSICAL METHODS: ISOTHERMAL TITRATION
CALORIMETRY AND SURFACE PLASMON RESONANCE SPECTROSCOPY 55 MIRCO GEISLER
TABLE OF CONTENTS 3.5 PROTEIN-PROTEIN INTERACTIONS IN A. THALIANA MOCO
BIOSYNTHESIS 56 4 MATERIAL AND METHODS 59 4.1 MATERIAL 59 4.1.1
CHEMICALS AND ENZYMES 59 4.1.2 EQUIPMENT 59 4.1.3 ORGANISM STRAINS 60
4.1.4 STANDARD MEDIA AND BUFFER 61 4.1.5 PLAMIDS 62 4.1.6
OLIGONUCLEOTIDES 63 4.2 METHODS 63 4.2.1 MOLECULAR BIOLOGICAL METHODS 63
4.2.1.1 PREPARATION OF PLASMID DNAFROM BACTERIA 63 4.2.1.2 RESTRICTION
ENDONUCLEASE DIGESTION OF DNA 63 4.2.1.3 LIGATION 64 4.2.1.4 DNA ELUTION
FROM AGAROSE GELS 64 4.2.1.5 GATEWAY CLONING 64 4.2.1.6 POLYMERASE CHAIN
REACTION (PCR) 65 4.2.1.7 PREPARATION OF COMPETENT . COLI 66 4.2.1.8
TRANSFORMATION INTO E. COLI 66 4.2.1.9 PREPARATION OF ELECTRO COMPETENT
A. TUMEFACIENS CELLS 66 4.2.1.10 TRANSFORMATION INTO A. TUMEFACIENS VIA
ELECTROPORATION 67 4.2.1.11 TRANSFORMATION INTO N. BENTHAMIANA 67 4.2.2
BIOCHEMICAL METHODS 68 4.2.2.1 OVEREXPRESSION OF RECOMBINANT PROTEINS IN
E.COLI 68 4.2.2.2 OVEREXPRESSION OF RECOMBINANT XDH AND AO PROTEINS IN
P. PASTORIS 68 4.2.2.3 AFFINITY PURIFICATION OF HIS-TAGGED PROTEIN BY
NI-NTA AGAROSE 69 4.2.2.4 AFFINITY PURIFICATION OF INTEIN-TAGGED PROTEIN
69 4.2.2.5 ANION EXCHANGE CHROMATOGRAPHY 70 4.2.2.6 BUFFER EXCHANGE OF
PROTEIN SOLUTIONS 70 4.2.2.7 DETERMINATION OF PROTEIN CONCENTRATIONS 70
4.2.2.8 SODIUM DODECYL SULFATE-POLYACRYLAMIDE GEL ELECTROPHORESIS
(SDS-PAGE) 71 4.2.2.9 SEMI DRY IMMUNOBLOT 71 MIRCO GEISLER TABLE OF
CONTENTS 4.3 ANALYTICAL METHODS 72 4.3.1 CROSSLINKING WITH BIOTIN LABEL
TRANSFER 72 4.3.1.1 INCORPORATION OF LINKER INTO A BAIT PROTEIN 73
4.3.1.2 CROSSLINKING AND DETECTION OF LABEL TRANSFER 73 4.3.2
PURIFICATION OF STREP-TAGGED PROTEINS FROM N. BENTHAMIANA 74 4.3.2.1
DETECTION OF STREP-TAGGED AND CO-PURIFIED PROTEINS FROM N. BENTHAMIANA
BY SILVER STAINING OF SDS-PA GELS 74 4.3.2.2 MASS SPECTROMETRY OF
CO-PURIFIED PROTEINS 75 4.3.3 GEL FILTRATION 75 4.3.4 SURFACE PLASMON
RESONANCE (SPR) SPECTROSCOPY 75 4.3.4.1 PROTOCOL FOR NON-COVALENT
CAPTURE OF HIS-TAGGED PROTEINS TO NTA CHIP 75 4.3.5 ISOTHERMAL TITRATION
CALORIMETRY 76 5 SUMMARY 77 ACKNOWLEDGEMENT 88
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| spelling | Geisler, Mirco Verfasser aut Protein-protein interactions in Arabidopsis thaliana molybdenum cofactor biosynthesis and allocation von Mirco Geisler 2010 88 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Braunschweig, Techn. Univ., Diss., 2009 (DE-588)4113937-9 Hochschulschrift gnd-content GBV Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=018942813&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Geisler, Mirco Protein-protein interactions in Arabidopsis thaliana molybdenum cofactor biosynthesis and allocation |
| subject_GND | (DE-588)4113937-9 |
| title | Protein-protein interactions in Arabidopsis thaliana molybdenum cofactor biosynthesis and allocation |
| title_auth | Protein-protein interactions in Arabidopsis thaliana molybdenum cofactor biosynthesis and allocation |
| title_exact_search | Protein-protein interactions in Arabidopsis thaliana molybdenum cofactor biosynthesis and allocation |
| title_full | Protein-protein interactions in Arabidopsis thaliana molybdenum cofactor biosynthesis and allocation von Mirco Geisler |
| title_fullStr | Protein-protein interactions in Arabidopsis thaliana molybdenum cofactor biosynthesis and allocation von Mirco Geisler |
| title_full_unstemmed | Protein-protein interactions in Arabidopsis thaliana molybdenum cofactor biosynthesis and allocation von Mirco Geisler |
| title_short | Protein-protein interactions in Arabidopsis thaliana molybdenum cofactor biosynthesis and allocation |
| title_sort | protein protein interactions in arabidopsis thaliana molybdenum cofactor biosynthesis and allocation |
| topic_facet | Hochschulschrift |
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