Snake venom proteins and their interaction with integrin α2β1- and GPlb-receptors:
Saved in:
| Main Author: | |
|---|---|
| Format: | Thesis Book |
| Language: | English |
| Published: |
Münster
[2021]
|
| Subjects: | |
| Online Access: | Inhaltsverzeichnis Inhaltsverzeichnis |
| Physical Description: | X, 125 Blätter Illustrationen, Diagramme 30 cm |
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MARC
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| 100 | 1 | |a Johanningmeier, Benjamin |d 1984- |e Verfasser |0 (DE-588)1251540074 |4 aut | |
| 245 | 1 | 0 | |a Snake venom proteins and their interaction with integrin α2β1- and GPlb-receptors |c vorgelegt von Benjamin Johanningmeier |
| 264 | 1 | |a Münster |c [2021] | |
| 300 | |a X, 125 Blätter |b Illustrationen, Diagramme |c 30 cm | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |b Dissertation |c Westfälische Wilhelms-Universität Münster |d 2022 |g Beschränkt für den Austausch | ||
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Record in the Search Index
| _version_ | 1804184938147217408 |
|---|---|
| adam_text | TABLE
OF
CONTENTS
LIST
OF
FIGURES
...........................................................................................................................................
IV
LIST
OF
TABLES
............................................................................................................................................
VI
LIST
OF
ABBREVIATIONS
...............................................................................................................................
VII
ABSTRACT
...................................................................................................................................................
IX
1.
INTRODUCTION
..........................................................................................................................................
1
1.1
VENOM
COMPOSITION
.......................................................................................................................
2
1.2
PLATELET
RECEPTORS
...........................................................................................................................
3
1.3
INTEGRIN
A201
..................................................................................................................................
4
1.4
C-TYPE
LECTIN-RELATED
PROTEINS
(CLRPS)
...........................................................................................
7
1.5
VON
WILLEBRAND
FACTOR
(VWF)
..........................................................................................................
8
1.6
GLYCOPROTEIN
IBA
(GPIBA)
.............................................................................................................
11
1.7
AIM
OF
THIS
THESIS
..........................................................................................................................
15
2.
MATERIALS
AND
METHODS
......................................................................................................................
16
2.1
MATERIALS
.......................................................................................................................................
16
2.1.1
CHEMICALS
...............................................................................................................................
16
2.1.2
LABORATORY
EQUIPMENT
...........................................................................................................
19
2.1.3
ANTIBIOTICS
..............................................................................................................................
21
2.1.4
BUFFER
AND
SOLUTIONS
..............................................................................................................
21
2.1.5
ANTIBODIES
..............................................................................................................................
24
2.1.6
CULTURE
MEDIA
........................................................................................................................
25
2.1.7
ORGANISMS
..............................................................................................................................
26
2.1.8
PLASMIDS
.................................................................................................................................
27
2.1.9
OLIGONUCLEOTIDES
....................................................................................................................
28
2.1.10
CHROMATOGRAPHIC
MATERIAL
..................................................................................................
29
2.1.11
OTHER
MATERIALS
...................................................................................................................
29
2.1.12
SOFTWARE
..............................................................................................................................
30
2.2
METHODS
.......................................................................................................................................
31
2.2.1
MOLECULAR
BIOLOGY
METHODS
................................................................................................
31
2.2.1.1
ISOLATION
OF
NUCLEIC
ACID
..................................................................................................
31
2.2.1.2
AGAROSE
GEL
ELECTROPHORESIS
...........................................................................................
31
2.2.1.3
POLYMERASE
CHAIN
REACTION
(PCR)
..................................................................................
31
2.2.1.4
RESTRICTION
ENZYMES
.......................................................................................................
32
2.2.1.5
LIGATION
...........................................................................................................................
33
2.2.1.6
GENERATING
COMPETENT
E.
COLI
CELLS
.................................................................................
33
TABLE
OF
CONTENT
2.2.1.7
HEAT
SHOCK
TRANSFORMATION
OF
E.
COLI
..............................................................................
33
2.2.1.8
QUANTIFICATION
OF
DNA
AND
DNA
SEQUENCING
.................................................................
34
2.2.2
SF-9
INSECT
CELLS:
CULTURE
AND
TRANSFECTION
............................................................................
34
2.2.2.1
CULTURING
SF-9
CELLS
..........................................................................................................
34
2.2.2.2
CRYOPRESERVATION
OF
SF-9
CELLS
........................................................................................
35
2.2.2.3
SF-9
CELL
LYSIS
....................................................................................................................
35
2.2.2.4
TRANSFECTION
AND
SUBCLONING
OF
SF-9
CELLS
.......................................................................
36
2.2.3
BIOCHEMICAL
METHODS
............................................................................................................
37
2.2.3.1
GENERAL
PROTEIN
METHODS
...............................................................................................
37
2.2.3.2
EXPRESSION
AND
PURIFICATION
OF
RECOMBINANT
GST-A2A
..................................................
39
2.2.3.3
PURIFICATION
OF
BOTHROPS
ALTERNATUS
VENOM
....................................................................
40
2.2.3.4
EXPRESSION
AND
PURIFICATION
OF
RECOMBINANT
VWF-AL
...................................................
42
2.2.3.5
PLATELET
AGGREGOMETRY
ASSAY
.........................................................................................
43
2.2.3.6
EXPRESSION
AND
PURIFICATION
OF
RECOMBINANT
GPIBA290
................................................
44
2.2.3.7
GLYCOCALICIN
PREPARATION
.................................................................................................
46
2.2.3.8
ELISA
...............................................................................................................................
49
3.
RESULTS
..............................................................................................................................................
53
3.1
INTEGRIN
A2A
DOMAIN
.....................................................................................................................
53
3.1.1
INTEGRIN
A2A
BINDING
SITE
INTERACTION
WITH
RHODOCETIN
.........................................................
53
3.1.1.1
LOCKED
OPEN
AND
CLOSED
A2A
CONFORMATIONS
.........................................................
53
3.1.1.2
MAPPING
THE
A2A
DOCKING-SITE
FOR
RC
............................................................................
55
3.1.2
INTEGRIN
A2A
BINDING
SITE
INTERACTION
WITH
BOTHROPS
ALTERNATUS
VENOM
FRACTIONS
..............
56
3.1.2.1
EXPRESSION
AND
PURIFICATION
OF
GST-A2A
.........................................................................
56
3.1.2.2
OPTIMAL
ELISA-ANTIBODY
CONCENTRATIONS
USING
CHECKERBOARD
TITRATION
........................
58
3.1.2.3
INTERACTION
STUDIES
OF
GST-A2A
WITH
COLLAGEN
1
..............................................................
59
3.1.2.4
IDENTIFICATION
OF
INTEGRIN
A2A
INHIBITORY
PROTEINS
FROM
B.
ALTERNATUS
SNAKE
VENOM
....
60
3.1.2.5
PURIFICATION
STRATEGY
OF
B.
ALTERNATUS
VENOM
.................................................................
61
3.1.2.6
INHIBITION
OF
GST-A2A-COLLAGEN
INTERACTION
BY
B.
ALTERNATUS
TOXIN
FRACTION
..................
64
3.1.2.7
PROTEIN-ANALYSIS
OF
POOL
#16-17
OF
8.
ALTERNATUS
VENOM
PROTEINS
................................
66
3.2
VON
WILLEBRAND
FACTOR-AL
............................................................................................................
69
3.2.1
ISOLATION
AND
PURIFICATION
OF
RECOMBINANT
VON
WILLEBRAND
FACTOR-AL
DOMAIN
....................
69
3.2.2
MS-ANALYSIS
OF
RVWF-AL
DOMAIN
...........................................................................................
71
3.2.3
ACTIVITY-TEST
OF
RVWF-AL
........................................................................................................
72
3.3
GLYCOPROTEIN
LB
A
..........................................................................................................................
73
3.3.1
CLONING
OF
GPIBA290
..............................................................................................................
73
3.3.2
TRANSFECTION
AND
EXPRESSION
OF
RECOMBINANT
GPIBA290......................................................
76
II
TABLE
OF
CONTENT
3.3.3
PURIFICATION
OF
GPIBA290
....................................................................................................
76
3.4
GLYCOCALICIN
...................................................................................................................................
81
3.4.1
ISOLATION
AND
PURIFICATION
OF
GLYCOCALICIN
...............................................................................
81
3.4.2
CAPTURING
GLYCOCALICIN
BY
ELISA
.............................................................................................
82
4.
DISCUSSION.........................................................................................................................................
84
4.1
INTEGRIN
A2A
-
RHODOCETIN
INTERACTION
..........................................................................................
84
4.2
INTEGRIN
A2A
DOMAIN
AND
BOTHROPS
ALTERNATUS
VENOM
PROTEINS
.................................................
88
4.3
GLYCOPROTEIN
LB
A
AND
VON
WILLEBRAND
FACTOR-AL
........................................................................
92
4.3.1
BACTERIAL
EXPRESSION
AND
ANALYSIS
OF
RECOMBINANT
VON
WILLEBRAND
FACTOR-AL
.....................
92
4.3.2
INSECT
CELL
EXPRESSION
AND
ANALYSIS
OF
RECOMBINANT
GPIBA290
(RGPIBA290)........................
94
REFERENCES..............................................................................................................................................
97
APPENDIX
...............................................................................................................................................
105
PUBLICATIONS
..........................................................................................................................................
123
ACKNOWLEDGEMENTS
..............................................................................................................................
124
CURRICULUM
VITAE
...................................................................................................................................
125
|
| adam_txt |
TABLE
OF
CONTENTS
LIST
OF
FIGURES
.
IV
LIST
OF
TABLES
.
VI
LIST
OF
ABBREVIATIONS
.
VII
ABSTRACT
.
IX
1.
INTRODUCTION
.
1
1.1
VENOM
COMPOSITION
.
2
1.2
PLATELET
RECEPTORS
.
3
1.3
INTEGRIN
A201
.
4
1.4
C-TYPE
LECTIN-RELATED
PROTEINS
(CLRPS)
.
7
1.5
VON
WILLEBRAND
FACTOR
(VWF)
.
8
1.6
GLYCOPROTEIN
IBA
(GPIBA)
.
11
1.7
AIM
OF
THIS
THESIS
.
15
2.
MATERIALS
AND
METHODS
.
16
2.1
MATERIALS
.
16
2.1.1
CHEMICALS
.
16
2.1.2
LABORATORY
EQUIPMENT
.
19
2.1.3
ANTIBIOTICS
.
21
2.1.4
BUFFER
AND
SOLUTIONS
.
21
2.1.5
ANTIBODIES
.
24
2.1.6
CULTURE
MEDIA
.
25
2.1.7
ORGANISMS
.
26
2.1.8
PLASMIDS
.
27
2.1.9
OLIGONUCLEOTIDES
.
28
2.1.10
CHROMATOGRAPHIC
MATERIAL
.
29
2.1.11
OTHER
MATERIALS
.
29
2.1.12
SOFTWARE
.
30
2.2
METHODS
.
31
2.2.1
MOLECULAR
BIOLOGY
METHODS
.
31
2.2.1.1
ISOLATION
OF
NUCLEIC
ACID
.
31
2.2.1.2
AGAROSE
GEL
ELECTROPHORESIS
.
31
2.2.1.3
POLYMERASE
CHAIN
REACTION
(PCR)
.
31
2.2.1.4
RESTRICTION
ENZYMES
.
32
2.2.1.5
LIGATION
.
33
2.2.1.6
GENERATING
COMPETENT
E.
COLI
CELLS
.
33
TABLE
OF
CONTENT
2.2.1.7
HEAT
SHOCK
TRANSFORMATION
OF
E.
COLI
.
33
2.2.1.8
QUANTIFICATION
OF
DNA
AND
DNA
SEQUENCING
.
34
2.2.2
SF-9
INSECT
CELLS:
CULTURE
AND
TRANSFECTION
.
34
2.2.2.1
CULTURING
SF-9
CELLS
.
34
2.2.2.2
CRYOPRESERVATION
OF
SF-9
CELLS
.
35
2.2.2.3
SF-9
CELL
LYSIS
.
35
2.2.2.4
TRANSFECTION
AND
SUBCLONING
OF
SF-9
CELLS
.
36
2.2.3
BIOCHEMICAL
METHODS
.
37
2.2.3.1
GENERAL
PROTEIN
METHODS
.
37
2.2.3.2
EXPRESSION
AND
PURIFICATION
OF
RECOMBINANT
GST-A2A
.
39
2.2.3.3
PURIFICATION
OF
BOTHROPS
ALTERNATUS
VENOM
.
40
2.2.3.4
EXPRESSION
AND
PURIFICATION
OF
RECOMBINANT
VWF-AL
.
42
2.2.3.5
PLATELET
AGGREGOMETRY
ASSAY
.
43
2.2.3.6
EXPRESSION
AND
PURIFICATION
OF
RECOMBINANT
GPIBA290
.
44
2.2.3.7
GLYCOCALICIN
PREPARATION
.
46
2.2.3.8
ELISA
.
49
3.
RESULTS
.
53
3.1
INTEGRIN
A2A
DOMAIN
.
53
3.1.1
INTEGRIN
A2A
BINDING
SITE
INTERACTION
WITH
RHODOCETIN
.
53
3.1.1.1
LOCKED
"OPEN"
AND
"CLOSED"
A2A
CONFORMATIONS
.
53
3.1.1.2
MAPPING
THE
A2A
DOCKING-SITE
FOR
RC
.
55
3.1.2
INTEGRIN
A2A
BINDING
SITE
INTERACTION
WITH
BOTHROPS
ALTERNATUS
VENOM
FRACTIONS
.
56
3.1.2.1
EXPRESSION
AND
PURIFICATION
OF
GST-A2A
.
56
3.1.2.2
OPTIMAL
ELISA-ANTIBODY
CONCENTRATIONS
USING
CHECKERBOARD
TITRATION
.
58
3.1.2.3
INTERACTION
STUDIES
OF
GST-A2A
WITH
COLLAGEN
1
.
59
3.1.2.4
IDENTIFICATION
OF
INTEGRIN
A2A
INHIBITORY
PROTEINS
FROM
B.
ALTERNATUS
SNAKE
VENOM
.
60
3.1.2.5
PURIFICATION
STRATEGY
OF
B.
ALTERNATUS
VENOM
.
61
3.1.2.6
INHIBITION
OF
GST-A2A-COLLAGEN
INTERACTION
BY
B.
ALTERNATUS
TOXIN
FRACTION
.
64
3.1.2.7
PROTEIN-ANALYSIS
OF
POOL
#16-17
OF
8.
ALTERNATUS
VENOM
PROTEINS
.
66
3.2
VON
WILLEBRAND
FACTOR-AL
.
69
3.2.1
ISOLATION
AND
PURIFICATION
OF
RECOMBINANT
VON
WILLEBRAND
FACTOR-AL
DOMAIN
.
69
3.2.2
MS-ANALYSIS
OF
RVWF-AL
DOMAIN
.
71
3.2.3
ACTIVITY-TEST
OF
RVWF-AL
.
72
3.3
GLYCOPROTEIN
LB
A
.
73
3.3.1
CLONING
OF
GPIBA290
.
73
3.3.2
TRANSFECTION
AND
EXPRESSION
OF
RECOMBINANT
GPIBA290.
76
II
TABLE
OF
CONTENT
3.3.3
PURIFICATION
OF
GPIBA290
.
76
3.4
GLYCOCALICIN
.
81
3.4.1
ISOLATION
AND
PURIFICATION
OF
GLYCOCALICIN
.
81
3.4.2
CAPTURING
GLYCOCALICIN
BY
ELISA
.
82
4.
DISCUSSION.
84
4.1
INTEGRIN
A2A
-
RHODOCETIN
INTERACTION
.
84
4.2
INTEGRIN
A2A
DOMAIN
AND
BOTHROPS
ALTERNATUS
VENOM
PROTEINS
.
88
4.3
GLYCOPROTEIN
LB
A
AND
VON
WILLEBRAND
FACTOR-AL
.
92
4.3.1
BACTERIAL
EXPRESSION
AND
ANALYSIS
OF
RECOMBINANT
VON
WILLEBRAND
FACTOR-AL
.
92
4.3.2
INSECT
CELL
EXPRESSION
AND
ANALYSIS
OF
RECOMBINANT
GPIBA290
(RGPIBA290).
94
REFERENCES.
97
APPENDIX
.
105
PUBLICATIONS
.
123
ACKNOWLEDGEMENTS
.
124
CURRICULUM
VITAE
.
125 |
| any_adam_object | 1 |
| any_adam_object_boolean | 1 |
| author | Johanningmeier, Benjamin 1984- |
| author_GND | (DE-588)1251540074 |
| author_facet | Johanningmeier, Benjamin 1984- |
| author_role | aut |
| author_sort | Johanningmeier, Benjamin 1984- |
| author_variant | b j bj |
| building | Verbundindex |
| bvnumber | BV048834146 |
| ctrlnum | (OCoLC)1338227413 (DE-599)DNB1251520332 |
| format | Thesis Book |
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| genre | (DE-588)4113937-9 Hochschulschrift gnd-content |
| genre_facet | Hochschulschrift |
| id | DE-604.BV048834146 |
| illustrated | Illustrated |
| index_date | 2024-07-03T21:36:02Z |
| indexdate | 2024-07-10T09:47:17Z |
| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-034099682 |
| oclc_num | 1338227413 |
| open_access_boolean | |
| owner | DE-355 DE-BY-UBR |
| owner_facet | DE-355 DE-BY-UBR |
| physical | X, 125 Blätter Illustrationen, Diagramme 30 cm |
| publishDate | 2021 |
| publishDateSearch | 2021 |
| publishDateSort | 2021 |
| record_format | marc |
| spelling | Johanningmeier, Benjamin 1984- Verfasser (DE-588)1251540074 aut Snake venom proteins and their interaction with integrin α2β1- and GPlb-receptors vorgelegt von Benjamin Johanningmeier Münster [2021] X, 125 Blätter Illustrationen, Diagramme 30 cm txt rdacontent n rdamedia nc rdacarrier Dissertation Westfälische Wilhelms-Universität Münster 2022 Beschränkt für den Austausch (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf https://d-nb.info/1251520332/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=034099682&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis 1\p emakn 0,05013 20220610 DE-101 https://d-nb.info/provenance/plan#emakn 2\p dnb 20220706 DE-101 https://d-nb.info/provenance/plan#dnb 3\p dnb 20220706 DE-101 https://d-nb.info/provenance/plan#dnb |
| spellingShingle | Johanningmeier, Benjamin 1984- Snake venom proteins and their interaction with integrin α2β1- and GPlb-receptors |
| subject_GND | (DE-588)4113937-9 |
| title | Snake venom proteins and their interaction with integrin α2β1- and GPlb-receptors |
| title_auth | Snake venom proteins and their interaction with integrin α2β1- and GPlb-receptors |
| title_exact_search | Snake venom proteins and their interaction with integrin α2β1- and GPlb-receptors |
| title_exact_search_txtP | Snake venom proteins and their interaction with integrin α2β1- and GPlb-receptors |
| title_full | Snake venom proteins and their interaction with integrin α2β1- and GPlb-receptors vorgelegt von Benjamin Johanningmeier |
| title_fullStr | Snake venom proteins and their interaction with integrin α2β1- and GPlb-receptors vorgelegt von Benjamin Johanningmeier |
| title_full_unstemmed | Snake venom proteins and their interaction with integrin α2β1- and GPlb-receptors vorgelegt von Benjamin Johanningmeier |
| title_short | Snake venom proteins and their interaction with integrin α2β1- and GPlb-receptors |
| title_sort | snake venom proteins and their interaction with integrin α2β1 and gplb receptors |
| topic_facet | Hochschulschrift |
| url | https://d-nb.info/1251520332/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=034099682&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT johanningmeierbenjamin snakevenomproteinsandtheirinteractionwithintegrina2b1andgplbreceptors |
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