Membrane protein expression and purification: screening methods for proteins of cellular Cholesterol Homeostasis
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| Format: | Thesis Book |
| Language: | English |
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16. November 2018
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| Physical Description: | XX Seiten, Seite 21-166, xxxiv Seiten Illustrationen 21 cm |
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| 100 | 1 | |a Bothe, Arne |d 1986- |e Verfasser |0 (DE-588)1177929546 |4 aut | |
| 245 | 1 | 0 | |a Membrane protein expression and purification |b screening methods for proteins of cellular Cholesterol Homeostasis |c Arne Bothe |
| 264 | 1 | |a Dortmund |c 16. November 2018 | |
| 300 | |a XX Seiten, Seite 21-166, xxxiv Seiten |b Illustrationen |c 21 cm | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |b Dissertation |c Technische Universität Dortmund | ||
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 856 | 4 | 2 | |m B:DE-101 |q application/pdf |u http://d-nb.info/1179391780/04 |3 Inhaltsverzeichnis |
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| adam_text | CONTENT
CONTENT
..........................................................................................................................................
IX
LIST
OF
FIGURES
...........................................................................................................................
XIV
LIST
OF
TABLES
...........................................................................................................................
XVII
ABBREVIATIONS
.......................................................................................................................
XVIII
1
ABSTRACT
&
ZUSAMMENFASSUNG
.........................................................................................
21
1.1
ABSTRACT
........................................................................................................
21
1.2
ZUSAMMENFASSUNG
.......................................................................................
23
2
INTRODUCTION
........................................................................................................................
25
2.1
CHOLESTEROL
.....................................................................................................
25
2.1.1
ATHEROSCLEROSIS
.....................................................................................
25
2.1.2
CHOLESTEROL
BIOSYNTHESIS
.......................................................................
27
2.1.3
HMG-COA
REDUCTASE
............................................................................
28
2.1.4
INHIBITION
OF
HMG-COA
REDUCTASE
BY
STATINS
......................................
30
2.1.5
SIDE
EFFECTS
OF
STATINS
.............................................................................
31
2.1.6
REGULATION
OF
HMG-COA
REDUCTASE
BY
AMP
KINASES
.........................
32
2.1.6
HMGCR
DEGRADATION
BY
ERAD
...........................................................
33
2.2
STEROL
HOMEOSTASIS
......................................................................................
34
2.2.1
THE
SREBP
PATHWAY
.............................................................................
34
2.2.1
INSIG
PROTEINS
........................................................................................
37
2.2.2
STEROL
BIOSYNTHESIS
IN
OTHER
DOMAINS
..................................................
38
2.3
ACCESSIBILITY
OF
MEMBRANE
PROTEIN
TO
STRUCTURAL
BIOLOGY
...........................
40
2.4
EXPRESSION
AND
FOLDING
OF
MEMBRANE
PROTEINS
..........................................
42
2.5
OBJECTIVE
......................................................................................................
44
3
MATERIAL
AND
METHODS
........................................................................................................46
3.1
MATERIAL
........................................................................................................
46
3.1.1
CHEMICALS
.............................................................................................
46
3.1.2
DEVICES
.................................................................................................
47
3.1.3
CELLS
AND
BACTERIAL
STRAINS
...................................................................
49
3.1.4
PLASMIDS
................................................................................................
49
3.1.5
OLIGONUCLEOTIDES
..................................................................................
49
3.1.6
ENZYMES
AND
PROTEINS
..........................................................................
49
3.2
MOLECULAR
BIOLOGY
.......................................................................................
50
3.2.1
DETERMINATION
OF
DNA
CONCENTRATIONS
................................................
50
3.2.2
RESTRICTION/LIGATION
CLONING
................................................................
50
3.2.2.1
POLYMERASE
CHAIN
REACTION
..............................................................
50
3.2.2.2
DIGESTION
OF
PLASMID
DNA
...............................................................
50
3.2.2.3
DIGESTION
OF
PCR
PRODUCTS
...............................................................
51
3.2.2.4
DNA
LIGATION
...................................................................................
51
3.2.2.5
RESTRICTION
ANALYSIS
..........................................................................
51
3.2.3
SLIC
CLONING
........................................................................................
52
3.2.3.1
POLYMERASE
CHAIN
REACTION
..............................................................
52
3.2.3.2
SLIC
REACTION
.....................................................................................
53
3.2.3.3
COLONY
PCR
.......................................................................................
53
3.2.4
SITE-DIRECTED
MUTAGENESIS
....................................................................
54
3.2.5
TRANSFORMATION
OF
CHEMICALLY
COMPETENT
CELLS
....................................
54
3.2.6
TRANSFORMATION
OF
ELECTRO-COMPETENT
E.
COLI
CELLS
...............................
54
3.2.7
SANGER
SEQUENCING
...............................................................................
55
3.3
PROTEIN
BIOCHEMISTRY
....................................................................................
55
3.3.1
SDS-PAGE
...........................................................................................
55
3.3.2
BLUE
NATIVE
PAGE
................................................................................
56
3.3.4 WESTERN
BLOT
.........................................................................................
56
3.3.5
TRYPTIC
IN-GEL
DIGESTION
........................................................................
57
3.3.6
HETEROLOGOUS
EXPRESSION
OF
PROTEINS
IN
E.
COLI
...................................
58
3.3.6.1
TRANSFORMATION
OF
E.
COLI
CELLS
.........................................................
58
33.6.2
EXPRESSION
SCREENING
IN
E.
COLI
.......................................................
58
3.3.7
HETEROLOGOUS
EXPRESSION
IN
S.
CEREVISIAE
...........................................
59
3.3.7.1
TRANSFORMATION
OF
S.
CEREVISIAE
CELLS
...............................................
59
33.1.2
HETEROLOGOUS
EXPRESSION
IN
S.
CEREVISIAE
........................................
60
3.3.8
HETEROLOGOUS
EXPRESSION
IN
P.
PASTOR
IS
..............................................
60
3.3.8.1
TRANSFORMATION
OF
P.
PASTORIS
...........................................................
60
3.3.8.2
EXPRESSION
ANALYSIS
IN
P.
PASTORIS
.....................................................
61
3.3.8.3
LARGE-SCALE
EXPRESSION
OF
PROTEINS
IN
P.
PASTORIS
.............................
63
3.3.9
INSECT
CELL
CULTURE
.................................................................................
64
3.3.9.1
BACULOVIRUS
PRODUCTION
MULTIBAC
AND
BACMAM
..............................
64
3.3.9.2
BACULOVIRUS
PRODUCTION
FLASHBAC
ULTRA
.....................................
66
33.9.3
EXPRESSION
TEST
IN
INSECT
CELLS
.........................................................
66
3.3.10
MAMMALIAN
CELL
CULTURE
........................................................................
67
X
3.3.10.1
ADHERENT
CELL
LINES
........................................................................
67
3.3.10.2
SUSPENSION
CELL
LINES
.....................................................................
68
3.3.10.3
HETEROLOGOUS
EXPRESSION
OF
PROTEINS
IN
MAMMALIAN
CELLS
.........
68
3.4
FLUORESCENCE-DETECTION
SIZE-EXCLUSION
CHROMATOGRAPHY
...........................
69
3.5
HIGH-THROUGHPUT
DETERGENT
SCREEN
..............................................................
70
3.5.1
PROTEIN
EXPRESSION
................................................................................
70
3.5.1.1
PATCHED
(
HOMO
SAPIENS)
...................................................................
70
3.5.1.2
HHAT
(HOMO
SAPIENS)
.....................................................................
70
3.5.1.3
HPNN
(
GEOBACTER
BEMIDJIENSIS)
......................................................
71
3.5.2
MEMBRANE
PREPARATION
.........................................................................
71
3.5.3
DETERGENT
SCREEN
..................................................................................
71
3.5.4
MANUAL
REPRODUCTION
...........................................................................
72
3.6
EXPRESSION
AND
PURIFICATION
..........................................................................
73
3.6.1
EXPRESSION
AND
PURIFICATION
OF
INSIG-
1
.................................................
73
3.6.2
EXPRESSION
AND
PURIFICATION
OF
EGFP-INSIGL
1-77
...............................
74
3.6.3
EXPRESSION
AND
PURIFICATION
OF
XTLNSIG-L
.............................................
75
3.6.4
EXPRESSION
OF
NSGLP
............................................................................
77
3.6.5
EXPRESSION
AND
PURIFICATION
OF
MCHERRY-HMGCR
..............................
77
3.6.6
EXPRESSION
OF
HMGCR
AND
HMGCR/INSIG-1
.....................................
78
3.6.7
EXPRESSION
AND
PURIFICATION
OF
MSP
PROTEINS
......................................
79
3.7
BIOPHYSICAL
METHODS
...................................................................................
80
3.7.1
UV/VIS
SPECTROSCOPY
...........................................................................
80
3.7.1.1
HMGCR
ACTIVITY
ASSAY
.....................................................................
80
3.7.2
CD
SPECTROSCOPY..................................................................................
80
3.7.3
MICROSCALE
THERMOPHORESIS..................................................................
81
3.7.4
NANODSF
..............................................................................................
82
3.7.5
H/D
EXCHANGE
MASS
SPECTROMETRY
......................................................
82
3.8
STRUCTURAL
BIOLOGY
METHODS
..........................................................................
83
3.8.1
NEGATIVE
STAIN
ELECTRON
MICROSCOPY
...................................................
83
3.8.2
SITTING
DROP
VAPOR
DIFFUSION
CRYSTALLIZATION......................................
84
3.8.3
LIPIDIC
CUBIC
PHASE
(ECP)
CRYSTALLIZATION
..........................................
84
4
RESULTS
...................................................................................................................................
85
4.1
INSULIN-INDUCED
GENES
.......................................................................................
85
4.1.1
EXPRESSION
AND
PURIFICATION
.................................................................
85
XI
4.1.2
STABILITY
ANALYSIS
OF
A7INSIG-L
BY
CD
SPECTROSCOPY
87
4.2
A
GENERALIZED
APPROACH
TOWARDS
MEMBRANE
PROTEIN
PURIFICATION
89
4.2.1
SELECTION
OF
GENES
AND
SUBCLONING
90
4.2.2
INITIAL
SCREEN
92
4.2.2.2 SCREENING
OF
STABLE
INSIG
VARIANTS
AND
CONSTRUCT
OPTIMIZATION
BY
FSEC
92
4.2.2.1
FSEC
SCREENING
OF
STABLE
HPNN
VARIANTS
100
4.2.3
EXPRESSION
SCREENING
AND
UPSCALING
101
4.2.3.1
EXPRESSION
SCREENING
OF
HUMAN
HMG-COA
REDUCTASE
101
4.2.4
DETERGENT
SCREEN
106
4.2.4.1
DEVELOPMENT
OF
A
HIGH-THROUGHPUT
DETERGENT
SCREEN
106
4.2.4.2 DETERGENT
SCREEN
OF
HMG-COA
REDUCTASE
110
4.2.4.3
SOLUBILIZATION
OF
OTHER
MEMBRANE
PROTEINS
116
4.2.5
BUFFER
OPTIMIZATION
BY
NANODSF
121
4.2.6 CONSTRUCT
OPTIMIZATION
BY
HDX
MASS
SPECTROMETRY
124
4.3
STRUCTURAL
AND
BIOPHYSICAL
STUDIES
OF
HMG-COA
REDUCTASE
128
4.3.1
PURIFICATION
AND
INITIAL
STRUCTURAL
ANALYSIS
OF
MCHERRY-HMGCR
128
4.3.2
CD
SPECTROSCOPIC
ANALYSIS
OF
RECOMBINANT
HMG-COA
REDUCTASE....
129
4.3.3
NEGATIVE
STAIN
ELECTRON
MICROSCOPY
OF
MCHERRY-HMG-COA
REDUCTASE
130
4.3.3
BIOPHYSICAL
STUDIES
OF
RECOMBINANT
HMG-COA
REDUCTASE
ACTIVITY...
134
4.3.3.1
IDENTIFICATION
OF
DETERGENTS
FOR
HMGCR
EXTRACTION
136
4.3.3.2
INFLUENCE
OF
STATINS
AND
MUTATIONS
ON
HMG-COA
REDUCTASE
ACTIVITY
139
4.3.3.3
STUDIES
OF
THE
HMG-COA
REDUCTASE/INSIG-
1
COMPLEX
141
4.3.4
INITIAL
PURIFICATION
OF
HMG-COA
REDUCTASE
145
4.4
EXPRESSION
OF
SOLUBLE
TRANSMEMBRANE
PROTEINS
147
4.4.1
MSP
FUSION
IN
A
MAMMALIAN
EXPRESSION
SYSTEM
148
4.4.2
MSP
FUSION
IN
AN
INSECT
CELL
EXPRESSION
SYSTEM
151
5
DISCUSSION
153
5.1
PURIFICATION
AND
CRYSTALLIZATION
OF
INSIG-
1
153
5.2
MEMBRANE
PROTEIN
SCREENING
153
5.2.1
SELECTION
OF
PROTEIN
SEQUENCES
154
5.2.2
FSEC
SCREENING
154
XII
5.2.3
PROTEIN
EXPRESSION
156
5.2.4
HTP-DETERGENT
SCREEN
156
5.2.5
BUFFER
AND
CONSTRUCT
OPTIMIZATION
160
5.3
HMG-COA
REDUCTASE
161
5.4
SOLUBLE
TRANSMEMBRANE
PROTEINS
164
6
SUMMARY
166
7
APPENDIX
I
7.1
LITERATURE
I
7.2
PLASMIDS
......................................................................................................
XIII
7.3
FSEC
TRACES
XIX
7.3.1
ASPERGILLUS
NIGER
INSIG
DOMAIN
PROTEIN
XIX
7.3.2
HOMO
SAPIENS
INSIG-
1
XX
7.3.3
HOMO
SAPIENS
INSIG-2
XXI
7.3.4
XENOPUS
SILURANA
TROPICALIS
INSIG-
1
XXII
7.3.5
XENOPUS
SILURANA
TROPICALIS
INSIG-2
XXIII
7.3.6
SACCHAROMYCES
CEREVISIAE
NSGLP
XXIV
7.3.7
OTHERS
XXV
7.4
HTP
DETERGENT
SCREEN
XXVI
7.4.1
HMGCR
XXVI
7.4.2
PATCHED-
1
XXVII
7.4.3
HPNN
XXVIII
7.4.4
CMNPCL
XXIX
7.4.5
INSIG-
1
XXX
7.4.6
HHAT
XXXI
7.4.7
PGRMC1
XXXII
7.4.8
NSGLP
XXXIII
7.4.9
NEGATIVE
CONTROL
XXXIV
XIII
|
| adam_txt |
CONTENT
CONTENT
.
IX
LIST
OF
FIGURES
.
XIV
LIST
OF
TABLES
.
XVII
ABBREVIATIONS
.
XVIII
1
ABSTRACT
&
ZUSAMMENFASSUNG
.
21
1.1
ABSTRACT
.
21
1.2
ZUSAMMENFASSUNG
.
23
2
INTRODUCTION
.
25
2.1
CHOLESTEROL
.
25
2.1.1
ATHEROSCLEROSIS
.
25
2.1.2
CHOLESTEROL
BIOSYNTHESIS
.
27
2.1.3
HMG-COA
REDUCTASE
.
28
2.1.4
INHIBITION
OF
HMG-COA
REDUCTASE
BY
STATINS
.
30
2.1.5
SIDE
EFFECTS
OF
STATINS
.
31
2.1.6
REGULATION
OF
HMG-COA
REDUCTASE
BY
AMP
KINASES
.
32
2.1.6
HMGCR
DEGRADATION
BY
ERAD
.
33
2.2
STEROL
HOMEOSTASIS
.
34
2.2.1
THE
SREBP
PATHWAY
.
34
2.2.1
INSIG
PROTEINS
.
37
2.2.2
STEROL
BIOSYNTHESIS
IN
OTHER
DOMAINS
.
38
2.3
ACCESSIBILITY
OF
MEMBRANE
PROTEIN
TO
STRUCTURAL
BIOLOGY
.
40
2.4
EXPRESSION
AND
FOLDING
OF
MEMBRANE
PROTEINS
.
42
2.5
OBJECTIVE
.
44
3
MATERIAL
AND
METHODS
.46
3.1
MATERIAL
.
46
3.1.1
CHEMICALS
.
46
3.1.2
DEVICES
.
47
3.1.3
CELLS
AND
BACTERIAL
STRAINS
.
49
3.1.4
PLASMIDS
.
49
3.1.5
OLIGONUCLEOTIDES
.
49
3.1.6
ENZYMES
AND
PROTEINS
.
49
3.2
MOLECULAR
BIOLOGY
.
50
3.2.1
DETERMINATION
OF
DNA
CONCENTRATIONS
.
50
3.2.2
RESTRICTION/LIGATION
CLONING
.
50
3.2.2.1
POLYMERASE
CHAIN
REACTION
.
50
3.2.2.2
DIGESTION
OF
PLASMID
DNA
.
50
3.2.2.3
DIGESTION
OF
PCR
PRODUCTS
.
51
3.2.2.4
DNA
LIGATION
.
51
3.2.2.5
RESTRICTION
ANALYSIS
.
51
3.2.3
SLIC
CLONING
.
52
3.2.3.1
POLYMERASE
CHAIN
REACTION
.
52
3.2.3.2
SLIC
REACTION
.
53
3.2.3.3
COLONY
PCR
.
53
3.2.4
SITE-DIRECTED
MUTAGENESIS
.
54
3.2.5
TRANSFORMATION
OF
CHEMICALLY
COMPETENT
CELLS
.
54
3.2.6
TRANSFORMATION
OF
ELECTRO-COMPETENT
E.
COLI
CELLS
.
54
3.2.7
SANGER
SEQUENCING
.
55
3.3
PROTEIN
BIOCHEMISTRY
.
55
3.3.1
SDS-PAGE
.
55
3.3.2
BLUE
NATIVE
PAGE
.
56
3.3.4 WESTERN
BLOT
.
56
3.3.5
TRYPTIC
IN-GEL
DIGESTION
.
57
3.3.6
HETEROLOGOUS
EXPRESSION
OF
PROTEINS
IN
E.
COLI
.
58
3.3.6.1
TRANSFORMATION
OF
E.
COLI
CELLS
.
58
33.6.2
EXPRESSION
SCREENING
IN
E.
COLI
.
58
3.3.7
HETEROLOGOUS
EXPRESSION
IN
S.
CEREVISIAE
.
59
3.3.7.1
TRANSFORMATION
OF
S.
CEREVISIAE
CELLS
.
59
33.1.2
HETEROLOGOUS
EXPRESSION
IN
S.
CEREVISIAE
.
60
3.3.8
HETEROLOGOUS
EXPRESSION
IN
P.
PASTOR
IS
.
60
3.3.8.1
TRANSFORMATION
OF
P.
PASTORIS
.
60
3.3.8.2
EXPRESSION
ANALYSIS
IN
P.
PASTORIS
.
61
3.3.8.3
LARGE-SCALE
EXPRESSION
OF
PROTEINS
IN
P.
PASTORIS
.
63
3.3.9
INSECT
CELL
CULTURE
.
64
3.3.9.1
BACULOVIRUS
PRODUCTION
MULTIBAC
AND
BACMAM
.
64
3.3.9.2
BACULOVIRUS
PRODUCTION
FLASHBAC
ULTRA
.
66
33.9.3
EXPRESSION
TEST
IN
INSECT
CELLS
.
66
3.3.10
MAMMALIAN
CELL
CULTURE
.
67
X
3.3.10.1
ADHERENT
CELL
LINES
.
67
3.3.10.2
SUSPENSION
CELL
LINES
.
68
3.3.10.3
HETEROLOGOUS
EXPRESSION
OF
PROTEINS
IN
MAMMALIAN
CELLS
.
68
3.4
FLUORESCENCE-DETECTION
SIZE-EXCLUSION
CHROMATOGRAPHY
.
69
3.5
HIGH-THROUGHPUT
DETERGENT
SCREEN
.
70
3.5.1
PROTEIN
EXPRESSION
.
70
3.5.1.1
PATCHED
(
HOMO
SAPIENS)
.
70
3.5.1.2
HHAT
(HOMO
SAPIENS)
.
70
3.5.1.3
HPNN
(
GEOBACTER
BEMIDJIENSIS)
.
71
3.5.2
MEMBRANE
PREPARATION
.
71
3.5.3
DETERGENT
SCREEN
.
71
3.5.4
MANUAL
REPRODUCTION
.
72
3.6
EXPRESSION
AND
PURIFICATION
.
73
3.6.1
EXPRESSION
AND
PURIFICATION
OF
INSIG-
1
.
73
3.6.2
EXPRESSION
AND
PURIFICATION
OF
EGFP-INSIGL
1-77
.
74
3.6.3
EXPRESSION
AND
PURIFICATION
OF
XTLNSIG-L
.
75
3.6.4
EXPRESSION
OF
NSGLP
.
77
3.6.5
EXPRESSION
AND
PURIFICATION
OF
MCHERRY-HMGCR
.
77
3.6.6
EXPRESSION
OF
HMGCR
AND
HMGCR/INSIG-1
.
78
3.6.7
EXPRESSION
AND
PURIFICATION
OF
MSP
PROTEINS
.
79
3.7
BIOPHYSICAL
METHODS
.
80
3.7.1
UV/VIS
SPECTROSCOPY
.
80
3.7.1.1
HMGCR
ACTIVITY
ASSAY
.
80
3.7.2
CD
SPECTROSCOPY.
80
3.7.3
MICROSCALE
THERMOPHORESIS.
81
3.7.4
NANODSF
.
82
3.7.5
H/D
EXCHANGE
MASS
SPECTROMETRY
.
82
3.8
STRUCTURAL
BIOLOGY
METHODS
.
83
3.8.1
NEGATIVE
STAIN
ELECTRON
MICROSCOPY
.
83
3.8.2
SITTING
DROP
VAPOR
DIFFUSION
CRYSTALLIZATION.
84
3.8.3
LIPIDIC
CUBIC
PHASE
(ECP)
CRYSTALLIZATION
.
84
4
RESULTS
.
85
4.1
INSULIN-INDUCED
GENES
.
85
4.1.1
EXPRESSION
AND
PURIFICATION
.
85
XI
4.1.2
STABILITY
ANALYSIS
OF
A7INSIG-L
BY
CD
SPECTROSCOPY
87
4.2
A
GENERALIZED
APPROACH
TOWARDS
MEMBRANE
PROTEIN
PURIFICATION
89
4.2.1
SELECTION
OF
GENES
AND
SUBCLONING
90
4.2.2
INITIAL
SCREEN
92
4.2.2.2 SCREENING
OF
STABLE
INSIG
VARIANTS
AND
CONSTRUCT
OPTIMIZATION
BY
FSEC
92
4.2.2.1
FSEC
SCREENING
OF
STABLE
HPNN
VARIANTS
100
4.2.3
EXPRESSION
SCREENING
AND
UPSCALING
101
4.2.3.1
EXPRESSION
SCREENING
OF
HUMAN
HMG-COA
REDUCTASE
101
4.2.4
DETERGENT
SCREEN
106
4.2.4.1
DEVELOPMENT
OF
A
HIGH-THROUGHPUT
DETERGENT
SCREEN
106
4.2.4.2 DETERGENT
SCREEN
OF
HMG-COA
REDUCTASE
110
4.2.4.3
SOLUBILIZATION
OF
OTHER
MEMBRANE
PROTEINS
116
4.2.5
BUFFER
OPTIMIZATION
BY
NANODSF
121
4.2.6 CONSTRUCT
OPTIMIZATION
BY
HDX
MASS
SPECTROMETRY
124
4.3
STRUCTURAL
AND
BIOPHYSICAL
STUDIES
OF
HMG-COA
REDUCTASE
128
4.3.1
PURIFICATION
AND
INITIAL
STRUCTURAL
ANALYSIS
OF
MCHERRY-HMGCR
128
4.3.2
CD
SPECTROSCOPIC
ANALYSIS
OF
RECOMBINANT
HMG-COA
REDUCTASE.
129
4.3.3
NEGATIVE
STAIN
ELECTRON
MICROSCOPY
OF
MCHERRY-HMG-COA
REDUCTASE
130
4.3.3
BIOPHYSICAL
STUDIES
OF
RECOMBINANT
HMG-COA
REDUCTASE
ACTIVITY.
134
4.3.3.1
IDENTIFICATION
OF
DETERGENTS
FOR
HMGCR
EXTRACTION
136
4.3.3.2
INFLUENCE
OF
STATINS
AND
MUTATIONS
ON
HMG-COA
REDUCTASE
ACTIVITY
139
4.3.3.3
STUDIES
OF
THE
HMG-COA
REDUCTASE/INSIG-
1
COMPLEX
141
4.3.4
INITIAL
PURIFICATION
OF
HMG-COA
REDUCTASE
145
4.4
EXPRESSION
OF
SOLUBLE
TRANSMEMBRANE
PROTEINS
147
4.4.1
MSP
FUSION
IN
A
MAMMALIAN
EXPRESSION
SYSTEM
148
4.4.2
MSP
FUSION
IN
AN
INSECT
CELL
EXPRESSION
SYSTEM
151
5
DISCUSSION
153
5.1
PURIFICATION
AND
CRYSTALLIZATION
OF
INSIG-
1
153
5.2
MEMBRANE
PROTEIN
SCREENING
153
5.2.1
SELECTION
OF
PROTEIN
SEQUENCES
154
5.2.2
FSEC
SCREENING
154
XII
5.2.3
PROTEIN
EXPRESSION
156
5.2.4
HTP-DETERGENT
SCREEN
156
5.2.5
BUFFER
AND
CONSTRUCT
OPTIMIZATION
160
5.3
HMG-COA
REDUCTASE
161
5.4
SOLUBLE
TRANSMEMBRANE
PROTEINS
164
6
SUMMARY
166
7
APPENDIX
I
7.1
LITERATURE
I
7.2
PLASMIDS
.
XIII
7.3
FSEC
TRACES
XIX
7.3.1
ASPERGILLUS
NIGER
INSIG
DOMAIN
PROTEIN
XIX
7.3.2
HOMO
SAPIENS
INSIG-
1
XX
7.3.3
HOMO
SAPIENS
INSIG-2
XXI
7.3.4
XENOPUS
SILURANA
TROPICALIS
INSIG-
1
XXII
7.3.5
XENOPUS
SILURANA
TROPICALIS
INSIG-2
XXIII
7.3.6
SACCHAROMYCES
CEREVISIAE
NSGLP
XXIV
7.3.7
OTHERS
XXV
7.4
HTP
DETERGENT
SCREEN
XXVI
7.4.1
HMGCR
XXVI
7.4.2
PATCHED-
1
XXVII
7.4.3
HPNN
XXVIII
7.4.4
CMNPCL
XXIX
7.4.5
INSIG-
1
XXX
7.4.6
HHAT
XXXI
7.4.7
PGRMC1
XXXII
7.4.8
NSGLP
XXXIII
7.4.9
NEGATIVE
CONTROL
XXXIV
XIII |
| any_adam_object | 1 |
| any_adam_object_boolean | 1 |
| author | Bothe, Arne 1986- |
| author_GND | (DE-588)1177929546 |
| author_facet | Bothe, Arne 1986- |
| author_role | aut |
| author_sort | Bothe, Arne 1986- |
| author_variant | a b ab |
| building | Verbundindex |
| bvnumber | BV046957032 |
| ctrlnum | (OCoLC)1189448162 (DE-599)DNB1179391780 |
| discipline | Biologie |
| discipline_str_mv | Biologie |
| format | Thesis Book |
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| genre | (DE-588)4113937-9 Hochschulschrift gnd-content |
| genre_facet | Hochschulschrift |
| id | DE-604.BV046957032 |
| illustrated | Illustrated |
| index_date | 2024-07-03T15:42:50Z |
| indexdate | 2024-07-10T08:58:34Z |
| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-032365454 |
| oclc_num | 1189448162 |
| open_access_boolean | |
| owner | DE-355 DE-BY-UBR |
| owner_facet | DE-355 DE-BY-UBR |
| physical | XX Seiten, Seite 21-166, xxxiv Seiten Illustrationen 21 cm |
| publishDate | 2018 |
| publishDateSearch | 2018 |
| publishDateSort | 2018 |
| record_format | marc |
| spelling | Bothe, Arne 1986- Verfasser (DE-588)1177929546 aut Membrane protein expression and purification screening methods for proteins of cellular Cholesterol Homeostasis Arne Bothe Dortmund 16. November 2018 XX Seiten, Seite 21-166, xxxiv Seiten Illustrationen 21 cm txt rdacontent n rdamedia nc rdacarrier Dissertation Technische Universität Dortmund (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf http://d-nb.info/1179391780/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=032365454&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Bothe, Arne 1986- Membrane protein expression and purification screening methods for proteins of cellular Cholesterol Homeostasis |
| subject_GND | (DE-588)4113937-9 |
| title | Membrane protein expression and purification screening methods for proteins of cellular Cholesterol Homeostasis |
| title_auth | Membrane protein expression and purification screening methods for proteins of cellular Cholesterol Homeostasis |
| title_exact_search | Membrane protein expression and purification screening methods for proteins of cellular Cholesterol Homeostasis |
| title_exact_search_txtP | Membrane protein expression and purification screening methods for proteins of cellular Cholesterol Homeostasis |
| title_full | Membrane protein expression and purification screening methods for proteins of cellular Cholesterol Homeostasis Arne Bothe |
| title_fullStr | Membrane protein expression and purification screening methods for proteins of cellular Cholesterol Homeostasis Arne Bothe |
| title_full_unstemmed | Membrane protein expression and purification screening methods for proteins of cellular Cholesterol Homeostasis Arne Bothe |
| title_short | Membrane protein expression and purification |
| title_sort | membrane protein expression and purification screening methods for proteins of cellular cholesterol homeostasis |
| title_sub | screening methods for proteins of cellular Cholesterol Homeostasis |
| topic_facet | Hochschulschrift |
| url | http://d-nb.info/1179391780/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=032365454&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT bothearne membraneproteinexpressionandpurificationscreeningmethodsforproteinsofcellularcholesterolhomeostasis |
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