Purification, folding, and kinetic analysis of individual disulfide bridge changed variants of the Bowman Birk inhibitor expressed in Escherichia coli:
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| Main Author: | |
|---|---|
| Format: | Thesis Book |
| Language: | German |
| Published: |
1994
|
| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | 114 S. Ill., graph. Darst. 21 cm |
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MARC
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| 100 | 1 | |a Park, Young-Mi |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Purification, folding, and kinetic analysis of individual disulfide bridge changed variants of the Bowman Birk inhibitor expressed in Escherichia coli |c by Young-Mi Park (Kim) |
| 264 | 1 | |c 1994 | |
| 300 | |a 114 S. |b Ill., graph. Darst. |c 21 cm | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |a Giessen, Univ., Diss., 1994 | ||
| 650 | 0 | 7 | |a Bowman-Birk-Inhibitor |0 (DE-588)4348202-8 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Struktur-Aktivitäts-Beziehung |0 (DE-588)4183784-8 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Sojabohne |0 (DE-588)4136938-5 |2 gnd |9 rswk-swf |
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| 689 | 0 | 0 | |a Sojabohne |0 (DE-588)4136938-5 |D s |
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CONTENTS
CONTENTS
PAGE
CONTENTS
OF
FIGURES
V
CONTENTS
OF
TABLES
VII
LIST
OF
ABBREVIATIONS
VIII
1
INTRODUCTION
1
2
REVIEW
OF
LITERATURE
3
2.1
PROTEIN
INHIBITORS
OF
PROTEASES
3
2.1.1
FOUR
CLASSES
OF
PROTEASE
INHIBITORS
3
2.1.2
INTERACTIONS
OF
PROTEIN
INHIBITORS
WITH
SERINE
PROTEASE
4
2.1.2.1
THE
STANDARD
MECHANISM
6
2.1.2.2
THE
REACTIVE
SITE
OF
INHIBITORS
7
2.1.2.3
DISULFIDE
BRIDGE
AND
REACTIVE
SITE
OF
INHIBITORS
10
2.1.2.4
REACTIVE
SITE
OF
SERINE
PROTEASE
12
2.2
THE
BOWMAN-BIRK
INHIBITOR
(
BBI
)
14
2.2.1
STRUCTURE
OF
BBI
15
2.2.2
REACTIVE
SITE
OF
BBI
18
2.2.3
DISULFIDE
BRIDGES
OF
BBI
21
3
MATERIALS
AND
METHODS
25
3.1
MATERIALS
25
3.1.1
INSTRUMENTS
25
3.1.2
MATERIALS
25
3.1.3
ENZYMES
26
3.1.4
CHEMICALS
26
3.2
METHODS
27
3.2.1
EXPRESSION
AND
ISOLATION
OF
BBI
VARIANTS
IN
E.
COH
27
3.2.1.1
CYANOGEN
BROMIDE
CLEAVAGE
OF
CRUDE
FUSION
PROTEIN
28
3.2.1.2
REDUCTION
AND
REOXIDATION
AFTER
CYANOGEN
BROMIDE
CLEAVAGE
28
CONTENTS
II
3.2.1.3
SODIUM
DODECYL
SULFATE-POLYACRYLAMIDE
GEL
ELECTROPHORESIS
(
SDS-PAGE
)
29
3.2.1.4
DETERMINATION
OF
PROTEIN
CONCENTRATION
30
3.2.1.5
PURIFICATION
OF
BBI
VARIANTS
BY
AFFINITY
CHROMATOGRAPHY
WITH
IMMOBILIZED
TRYPSIN
(
TRYPSIN-SEPHAROSE
)
30
3.2.1.5.1
PURIFICATION
OF
BBI
VARIANTS
30
3.2.1.5.2
DETECTION
OF
BBI
VARIANTS
31
3.2.2
KINETIC
ASSAYS
OF
BBI
VARIANTS
32
3.2.2.1
DETERMINATION
OF
TRYPSIN
AND
CHYMOTRYPSIN-ACTIVE
SITE
CONCENTRATION
32
3.2.2.2
DETERMINATION
OF
RESIDUAL
CHYMOTRYPSIN
ACTIVITY
32
3.2.2.3
DETERMINATION
OF
THE
APPARENT
EQUILIBRIUM
DISSOCIATION
33
3.2.2.4
CONSTANT
(
^I(APP)
)
DETERMINATION
OR
THE
ASSOCIATION
RATE
CONSTANT
(
K
OW
)
34
3.2.2.5
DETERMINATION
OF
THE
DISSOCIATION
CONSTANT
(
AND
^OFF)
DETERMINATION
OF
THE
DISSOCIATION
CONSTANT
WITHOUT
34
3.2.2.5.1
35
CARBOXYPEPTIDASE
B
(
K
O
FF)
DETERMINATION
OF
THE
DISSOCIATION
CONSTANT
WITH
3.2.2.5.2
35
CARBOXYPEPTIDASE
B
(
LY
WJ
)
3.2.3
PURIFICATION
OF
R.BBI
BY
AFFINITY
CHROMATOGRAPHY
WITH
IMMOBILIZED
ANHYDROTRYPSIN
35
3.2.3.1
PREPARATION
AND
PURIFICATION
OF
ANHYDROTRYPSIN
35
3.2.3.2
PURIFICATION
OF
R.BBI
BY
AFFINITY
CHROMATOGRAPHY
WITH
IMMOBILIZED
ANHYDROTRYPSIN
36
3.2.4
PURIFICATION
OF
RBBI
WITH
ION
EXCHANGE
CHROMATOGRAPHIES
AND
37
REVERSED-PHASE
HIGH
PRESSURE
LIQUID
CHROMATOGRAPHY
(
HPLC
)
3.2.4.1
PURIFICATION
OF
R.BBI
ON
DEAE-SEPHAROSE
37
3.2.4.2
PURIFICATION
OF
R.BBI
WITH
CATION
EXCHANGE
CHROMATOGRAPHY
USING
FRACTO
GEL
TSK
SP-650
(S)
37
3.2.4.3
PURIFICATION
OF
R.BBI
WITH
ANION
EXCHANGE
CHROMATOGRAPHY
USING
TMAE
650
(M)
38
3.2.4.4
REVERSED-PHASE
HPLC
38
3.2.5
PREPARATION,
ISOLATION
AND
KINETIC
ANALYSIS
OF
MODIFIED
BBI
(
BBI*
)
39
3.2.5.1
PREPARATION
OF
BBI*
39
3.2.5.2
ISOLATION
OF
BBI*
BY
TRICINE-SODIUM
DODECYL
SULFATE
POLYACRYLAMIDE
GEL
ELECTROPHORESIS
(
TSDS-PAGE
)
39
3.2.5.2.1
TSDS-PAGE
39
3.2.5.2.2
PROCEDURE
FOR
SILVER
STAINING
POLYACRYLAMIDE
GELS
41
3.2.5.3
KINETIC
ASSAYS
OF
BBI*
WITH
TRYPSIN
41
3.2.5.3.1
DETERMINATION
OF
THE
EQUILIBRIUM
DISSOCIATION
CONSTANT
(
K(
*
)
41
3.2.5.3.2
DETERMINATION
OF
THE
ASSOCIATION
RATE
CONSTANT
(
K
ORT
*
)
42
3.2.S.3.3
RESYNTHESIS
OF
BBI
FROM
BBI*
42
3.2.6
PREPARATION
OF
MODIFIED
R.BBI*
42
CONTENTS
III
4
RESULTS
43
4.1
EXPRESSION
OF
BBI
VARIANTS
43
4.1.1
4.1.2
4.1.3
4.1.3.1
4.1.3.2
ENRICHMENT
OF
FUSION
PROTEINS
44
CLEAVAGE
OF
FUSION
PROTEIN
WITH
BRCN
46
REDUCTION
AND
REOXIDATION
46
REOXIDATION
CONDITIONS
47
REDUCTION
AND
REOXIDATION
OF
BBI
VARIANTS
AND
PLZPWBL
48
4.2
AFFINITY
CHROMATOGRAPHY
WITH
IMMOBILIZED
TRYPSIN
(
TRYPSIN
52
SEPHAROSE
)
4.2.1
ACTIVATION
AND
PURIFICATION
OF
BBI
VARIANTS
USING
AFFINITY
53
CHROMATOGRAPHY
WITH
IMMOBILIZED
TRYPSIN
4.3
KINETIC
ANALYSIS
OF
BBI
VARIANTS
55
4.3.1
APPARENT
EQUILIBRIUM
DISSOCIATION
CONSTANTS
AND
EQUILIBRIUM
55
DISSOCIATION
CONSTANTS
(
^-IFAPP)
AND
K,
)
4.3.2
4.3.3
4.3.3.1
4.3.3.2
4.3.4
ASSOCIATION
RATE
CONSTANTS
(
K
0
YY
)
58
DISSOCIATION
RATE
CONSTANTS
(
K
O
-AND
K^,
SS
)
61
DISSOCIATION
RATE
CONSTANTS
WITHOUT
CARBOXYPEPTIDASE
B
(
K^)
61
DISSOCIATION
RATE
CONSTANTS
WITH
CARBOXYPEPTIDASE
B
(
K^
M
)
65
BIMOLECULAR
DISSOCIATION
RATE
CONSTANTS
(
K
C
=
K
O
FF/K
ON
)
67
4.4
AFFINITY
CHROMATOGRAPHY
WITH
IMMOBILIZED
ANHYDROTRYPSIN
FOR
68
COMPLEMENTARY
REFOLDING
AND
PURIFICATION
4.5
PURIFICATION
OF
R.BBI
WITH
ION
EXCHANGE
CHROMATOGRAPHIES
AND
69
REVERSED-PHASE
HPLC
AND
IT'S
KINETIC
ANALYSIS
4.5.1
PURIFICATION
OF
R.BBI
WITH
ANION
EXCHANGE
CHROMATOGRAPHY
70
USING
A
DEAE-SEPHAROSE
4.5.2
PURIFICATION
OF
R.BBI
WITH
CATION
EXCHANGE
CHROMATOGRAPHY
71
USING
A
FRACTOGEL
TSK
SP-650
(S)
AND
REVERSED-PHASE
HPLC
4.5.3
PURIFICATION
OF
R.BBI
WITH
ANION
EXCHANGE
CHROMATOGRAPHY
72
USING
A
TMAE
650
(M)
AND
REVERSED-PHASE
HPLC
4.5.4
YIELD
AND
KINETIC
ANALYSIS
OF
R.BBI
74
4.6
PREPARATION,
ISOLATION
AND
KINETIC
ANALYSIS
OF
BBI*
77
4.6.1
4.6.2
4.6.3
PREPARATION
OF
BBI*
77
ISOLATION
OF
BBI*
78
KINETIC
ANALYSIS
OF
BBI*
79
CONTENTS
IV
4.7
PREPARATION
OF
R.BBI*
81
5
DISCUSSION
82
5.1
EXPRESSION
AND
ENRICHMENT
OF
FUSION
PROTEINS
82
5.2
REFOLDING
83
5.3
KINETIC
ANALYSIS
84
5.3.1
5.3.2
5.3.2.1
5.3.2.2
5.3.2.3
5.3.2.4
5.3.2.5
5.3.3
KINETIC
ANALYSIS
OF
R.BBI
85
KINETIC
ANALYSIS
OF
BBI
VARIANTS
86
KINETIC
ANALYSIS
OF
ID1
87
KINETIC
ANALYSIS
OF
ID2
87
KINETIC
ANALYSIS
OF
ID3
87
KINETIC
ANALYSIS
OF
ID4
88
KINETIC
ANALYSIS
OF
ID5
88
BBI
VARIANTS
AND
BBI
DISPLAY
THREE
KINETIC
MECHANISMS
89
5.4
HYDROLYSIS
91
6
CONCLUSION
93
7
SUMMARY
94
8
ZUSAMMENFASSUNG
96
9
REFERENCES
98 |
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| genre_facet | Hochschulschrift |
| id | DE-604.BV035191795 |
| illustrated | Illustrated |
| index_date | 2024-07-02T23:01:52Z |
| indexdate | 2024-08-16T00:43:39Z |
| institution | BVB |
| language | German |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-016998389 |
| oclc_num | 257767666 |
| open_access_boolean | |
| owner | DE-11 |
| owner_facet | DE-11 |
| physical | 114 S. Ill., graph. Darst. 21 cm |
| publishDate | 1994 |
| publishDateSearch | 1994 |
| publishDateSort | 1994 |
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| spelling | Park, Young-Mi Verfasser aut Purification, folding, and kinetic analysis of individual disulfide bridge changed variants of the Bowman Birk inhibitor expressed in Escherichia coli by Young-Mi Park (Kim) 1994 114 S. Ill., graph. Darst. 21 cm txt rdacontent n rdamedia nc rdacarrier Giessen, Univ., Diss., 1994 Bowman-Birk-Inhibitor (DE-588)4348202-8 gnd rswk-swf Struktur-Aktivitäts-Beziehung (DE-588)4183784-8 gnd rswk-swf Sojabohne (DE-588)4136938-5 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Sojabohne (DE-588)4136938-5 s Bowman-Birk-Inhibitor (DE-588)4348202-8 s Struktur-Aktivitäts-Beziehung (DE-588)4183784-8 s DE-604 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=016998389&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Park, Young-Mi Purification, folding, and kinetic analysis of individual disulfide bridge changed variants of the Bowman Birk inhibitor expressed in Escherichia coli Bowman-Birk-Inhibitor (DE-588)4348202-8 gnd Struktur-Aktivitäts-Beziehung (DE-588)4183784-8 gnd Sojabohne (DE-588)4136938-5 gnd |
| subject_GND | (DE-588)4348202-8 (DE-588)4183784-8 (DE-588)4136938-5 (DE-588)4113937-9 |
| title | Purification, folding, and kinetic analysis of individual disulfide bridge changed variants of the Bowman Birk inhibitor expressed in Escherichia coli |
| title_auth | Purification, folding, and kinetic analysis of individual disulfide bridge changed variants of the Bowman Birk inhibitor expressed in Escherichia coli |
| title_exact_search | Purification, folding, and kinetic analysis of individual disulfide bridge changed variants of the Bowman Birk inhibitor expressed in Escherichia coli |
| title_exact_search_txtP | Purification, folding, and kinetic analysis of individual disulfide bridge changed variants of the Bowman Birk inhibitor expressed in Escherichia coli |
| title_full | Purification, folding, and kinetic analysis of individual disulfide bridge changed variants of the Bowman Birk inhibitor expressed in Escherichia coli by Young-Mi Park (Kim) |
| title_fullStr | Purification, folding, and kinetic analysis of individual disulfide bridge changed variants of the Bowman Birk inhibitor expressed in Escherichia coli by Young-Mi Park (Kim) |
| title_full_unstemmed | Purification, folding, and kinetic analysis of individual disulfide bridge changed variants of the Bowman Birk inhibitor expressed in Escherichia coli by Young-Mi Park (Kim) |
| title_short | Purification, folding, and kinetic analysis of individual disulfide bridge changed variants of the Bowman Birk inhibitor expressed in Escherichia coli |
| title_sort | purification folding and kinetic analysis of individual disulfide bridge changed variants of the bowman birk inhibitor expressed in escherichia coli |
| topic | Bowman-Birk-Inhibitor (DE-588)4348202-8 gnd Struktur-Aktivitäts-Beziehung (DE-588)4183784-8 gnd Sojabohne (DE-588)4136938-5 gnd |
| topic_facet | Bowman-Birk-Inhibitor Struktur-Aktivitäts-Beziehung Sojabohne Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=016998389&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT parkyoungmi purificationfoldingandkineticanalysisofindividualdisulfidebridgechangedvariantsofthebowmanbirkinhibitorexpressedinescherichiacoli |