Cell-free protein synthesis: methods and protocols
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Wiley-VCH
2008
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| ISBN: | 9783527316496 3527316493 |
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| 020 | |a 3527316493 |c Gb. : ca. EUR 89.00 (freier Pr.), ca. sfr 142.00 (freier Pr.) |9 3-527-31649-3 | ||
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| 245 | 1 | 0 | |a Cell-free protein synthesis |b methods and protocols |c ed. by Alexander S. Spirin ... |
| 246 | 1 | 3 | |a Cell free protein synthesis |
| 264 | 1 | |a Weinheim |b Wiley-VCH |c 2008 | |
| 300 | |a XX, 242 S. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 650 | 4 | |a Cell-Free System |x physiology | |
| 650 | 4 | |a Genetic translation | |
| 650 | 4 | |a Protein Biosynthesis | |
| 650 | 4 | |a Proteins |x Synthesis | |
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| 856 | 4 | 2 | |q text/html |u http://deposit.dnb.de/cgi-bin/dokserv?id=2878010&prov=M&dok_var=1&dok_ext=htm |3 Inhaltstext |
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Datensatz im Suchindex
| _version_ | 1805088618133323776 |
|---|---|
| adam_text |
Contents
Preface XIII
List of Contributors
ХУЯ
1
Cell-free Protein Synthesis Systems: Historical Landmarks,
Classification, and General Methods
1
A. S. Spirin and]. R. Swartz
1.1
Introduction: Historical Landmarks
1
1.1.1
Discovery of Protein Synthesis in Cell Extracts
1
1.1.2
Translation of Exogenous Messages
1
1.1.3
Coupled Transcription-translation in Bacterial Extracts
2
1.1.4
Combined Transcription-translation Systems
3
1.1.5
Continuous Flow/Continuous Exchange Principle
3
1.2
Prokaryotic and Eukaryotic Types of Cell-free Expression
Systems
5
1.2.1
Cell Extracts
5
1.2.1.1
E. coli
extract
(ЕСЕ)
5
1.2.1.2
Wheat Germ Extract (WGE)
6
1.2.1.3
Rabbit Reticulocyte
Lysáte
(RRL)
6
1.2.2
Genetic Constructs (Expression Vectors)
7
1.2.2.1
Prokaryotic Systems
7
1.2.2.2
Eukaryotic Systems
8
1.3
Preparing Cell Extracts
11
1.3.1
E. coli
Extracts
11
1.3.1.1
Genetics
11
1.3.1.2
Cell Growth
13
1.3.1.3
Extract Preparation
14
1.3.2
Wheat Germ Extracts
15
1.4
Designing Reaction Composition
16
1.4.1
Mg2+ and Phosphate
16
1.4.2
Other Salts
18
1.4.3
Nucleotides and
Amino
Acids
18
1.4.4
Stabilization Reagents
ÍS
Cell-free Protein Synthesis. Edited by Alexander Spirin and James Swartz
Copyright
© 2008
WILEY-VCH
Verlag
GmbH
&
Co. KGaA,
Weinheim
ISBN:
978-3-527-31649-6
VI
I Contents
1.4.5
Other Factors
19
1.5
Providing Energy
19
1.5.1
Direct Nucleotide Regeneration
20
1.5.2
Indirect Nucleotide Regeneration
20
1.6
Enhancing Protein Folding
21
1.6.1
Temperature Effects
21
1.6.2
Cell Extract Concentration
23
1.6.3
Effects of Folding Ligands
23
1.6.4
Effects of Chaperones and Foldases
24
1.6.5
Effects of Detergents
24
2
The Constructive Approach for Cell-free Translation
35
T. Ueda
2.1
Introduction
35
2.2
The Process of Protein Synthesis
36
2.2.1
Polypeptide Synthesis
36
2.2.2
Protein Maturation
38
2.3
A Constructive Approach to Protein Synthesis
40
2.3.1
In Vitro
Reconstitution
of Polypeptide Synthesis
40
2.3.2
Protocol of Protein Synthesis using PURE System
41
2.3.3
Addition of Protein Folding Machinery to the PURE System
42
2.3.4
Integration of a Membrane Targeting System with the PURE
system
46
2.3.5
Protein Synthesis using the PURE System containing Molecular
Chaperones
48
2.4
Conclusion
49
3
Functional Cenomic Analysis using Sequential Cell-free Protein
Synthesis
51
K. A. Woodrow and]. R. Swartz
3.1
Introduction
5Î
3.1.1
The Post-genomic Era
51
3.1.2
Cell-free Protein Synthesis (CFPS) as a Functional Proteomic
Tool
52
3.2
Developing an enabling Technology for Sequential Expression
Analysis
54
3.2.1
Improving Linear Template Stability
55
3.2.2
Improving PCR Reactions for generating Genomic Linear
Templates
56
3.2.3
Optimizing Cofactor Concentrations for Enzyme Activation
58
3.3
Demonstrating Functional Genomic Analysis with CFPS
61
3.3.1
Isolation and Expression of Genomic Targets
62
Contents
VII
3.3.2
Effects of Sample Library on
ß-Lactamase
Expression and
Activity
62
3.4
Conclusions and Projections
64
4
Cell-free Technology for Rapid Production of Patient-specific Fusion
Protein Vaccines
69
A. R. Gotrke.,]. Yang, G.
Kanter,
R.
Levy and
J. R.
Swartz
4.1
Introduction
69
4.1.1
Lymphoma and Fusion Protein Vaccine Treatments
69
4.1.2
Comparing Cell-free and In Vivo Production Systems
70
4.2
Developing the Fusion Protein Construct and the Cell-free
Production Process
71
4.2.1
Fusion-protein Production in the Cell-free System
71
4.2.2
Oxidized Reaction Conditions and DsbC Increase Soluble Protein
Yield
71
4.2.3
GM-CSF is more Active at the N-terminus of the Fusion Protein
Vaccine
73
4.2.4
New Linker Improves Fusion Protein Stability
75
4.2.5
Expression and Purification Scale-up for Vaccine Protein
Production
77
4.3
Fusion Proteins Raise Protective Antibodies
78
4.3.1
Design of Vaccine Constructs and Mouse Studies
78
4.3.2
Fusion Protein Vaccination Protects against Aggressive Tumors
79
4.3.3
Antibody Generation is enhanced by Fusion Partners
79
4.4
Conclusions and Projections
80
5
Bacterial Cell-free System for Highly Efficient Protein Synthesis
83
T. Kigawa, T. Matsuda, T. Yabuki and S. Yokoyama
5.1
Overview
83
5.2
Introduction
83
5.3
Coupled Transcription-Translation System based on
E. coli
Extract
84
5.4 DNA
Template Construction
84
5.5
Preparation of Cell Extract from
E. coli
85
5.6
Batch-mode Cell-free Reaction
87
5.7
Dialysis-mode Cell-free Reaction
88
5.8
Template
DNA 91
5.9
Reaction Temperature
92
5.10
Surface Area of the Dialysis Membrane
93
5.11
Stable-isotope Labeling for NMR Spectroscopy
93
5.12
Selenomethionine Incorporation for
Х
-Ray Crystallography
94
5.13
Automation
95
5.14
Conclusion
95
VIII Contents
б
The Use of the
Escherichìa coli
Cell-free Protein Synthesis for
Structural Biology and Structural Proteomics
99
T. Kigawa, M. Inoue, M. Aoki, T. Matsuda, T. Yahuki, E. Seki,
T. Harada, S. Watanabe and S. Yokoyama
6.1
Overview
99
6.2
Introduction
100
6.3
High-throughput Expression by PCR-based Small-scale Cell-free
Protein Synthesis
100
6.4
Fully Automated Protein Production using Middle-scale Cell-free
Protein Synthesis
203
6.5
NMR Screening
104
6.6
Large-scale Protein Production for Structure Determination
105
6.7
Discussion
107
7
The Wheat Germ Cell-free Protein Synthesis System 111
T. Sawasaki and Y. Endo
7.1
Overview 111
7.2
Development of a Highly Efficient Eukaryotic Cell-free Protein
Synthesis System 111
7.2.1
Preparation of a Highly Active and Robust Extract from Wheat
Embryos
112
7.2.1.1
Protocol for the Preparation of Wheat Germ Extract
[12] 115
7.2.2
mRNA
5'
and
3'
UTRs which Enhance Translation
115
7.2.3
Split-primer PCR for Genome-wide Generation of DNAs for
Transcription
119
7.2.3.1
Protocol for "Split-primer" PCR
[13] 121
First PCR
122
7.2.4
Bilayer Translation Reaction Method
122
7.2.5
Transcription and Translation in One Tube
123
7.2.5.1
Protocol for One-tube Protein Synthesis Reaction
124
7.2.6
Reaction Methods for Large-scale Protein Production
125
7.3
Completion of Protocols for the Wheat Cell-free System
126
7.3.1
Performance of the Wheat Cell-free System
127
7.3.2
Robotic Automation of the Cell-free Protein Synthesis
132
7.4
Application to High-throughput Biochemical Annotation of Genetic
Information
132
7.4.1
Genome-wide Functional Analysis
132
7.4.2
Preparation of Protein for NMR Spectroscopy
134
7.5
Conclusion
136
Contents
IX
8
Cell-free
Expression
of
Integral Membrane Proteins
for Structural
Studies
141
С.
Klammt,
D.
Schwarz,
I.
Lehner,
S.
Sobhanifar,
F. Uhr,
J. Zeelen,
C. Glaubitz,
V.
Dötsch and F. Bernhard
8.1
Overview
141
8.2
Introduction
141
8.3
Specific Characteristics for the Cell-free Expression of Membrane
Proteins
143
8.3.1
Cell-free Expression of Membrane Proteins in the Presence of
Detergents or Lipids
145
8.3.2
Detergents for the Efficient Resolubilization of Cell-free Produced
Membrane Proteins
149
8.4
Case Studies for the High Level Cell-free Expression of Membrane
Proteins
150
8.4.1
a-Helical Transporters
150
8.4.2
G-Protein Coupled Receptors
252
8.4.3
^-Barrel Proteins
153
8.5
Structural Characterization of Cell-free Produced Membrane
Proteins
154
8.5.1
Crystallization of Cell-free Produced Membrane Proteins
154
8.5.2
Cell-free Expression as a Tool for High-resolution NMR
Spectroscopy
155
8.5.3
Applications of Cell-free Expression for Solid-state NMR
159
9
Cell-free Production of Membrane Proteins in the Presence of
Detergents
165
J.-M. Betton and M.
Miot
9.1
Introduction
165
9.2
Histidine Protein Kinases
166
9.3
Materials and Methods
168
9.3.1
Plasmids
368
9.3.2
Cell-free Protein Production
168
9.3.3
Protein Purification
168
9.3.4
Structural and Functional Protein Characterizations
169
9.4
Results and Discussion
169
9.4.1
Analytical Cell-free Production of
HÍS6
-tagged Proteins
169
9.4.2
Detergents Compatible with Cell-free Synthesis
171
9.4.3
Fidelity of In Vitro Biosynthesis Reactions in the Presence of
Brij35
173
9.4.4
High-level Production of Functional HPKs in CECF Technology
174
9.5
Conclusions
177
X I Contents
10
Novel Techniques using PCR and Cell-free Protein Synthesis Systems
for Combinatorial
Bioengineering 179
Η.
Nakano
and
T. Yamane
10.1
Introduction
379
10.2
Improvements in the
Escherìchia coli
Cell-free Protein Synthesis
Systems
180
10.3
High-throughput Construction of a Protein Library by SIMPLEX
180
10.3.1
Development of SIMPLEX
180
10.3.2
Quality of the SIMPLEX-based Protein Library
182
10.3.3
Expansion of the SIMPLEX-based Library
182
10.3.4
Application of SIMPLEX for Combinatorial Engineering of
Proteins
184
10.4
Development and Application of
SICREX
186
10.5
Conclusion
188
Π
Gene Cloning and Expression in Molecular Colonies
191
A. B.
Chetverìn,
T. R.
Samalov and
H. V. Chetverina
11.1
A Gap in Cell-free Biotechnology 1
91
11.2
Molecular Colony Technique
292
11.3
Gene Cloning in Molecular Colonies
193
11.4
Gene Expression in Molecular Colonies: Transcription
196
11.5
Gene Expression in Molecular Colonies: Translation
196
11.6
Gene Expression in Molecular Colonies: The Role of Thiol
Compounds
198
11.7
Conclusions
200
11.8
Molecular Colony Protocols
201
11.8.1
Amplification Gels
201
11.8.2
Growing
DNA
Colonies
202
11.8.3
Detection of Molecular Colonies
202
11.8.4
Transcription in Molecular Colonies
203
11.8.5
Protein Synthesis in Molecular Colonies
203
12
Large-Scale Batch Reactions for Cell-free Protein Synthesis
207
A. M. Voloshin and]. R. Swartz
12.1
Introduction
207
12.1.1
Cell-free Protein Synthesis
207
12.1.2
Comparing Cell-free Reaction Configurations; Advantages of Batch
Mode
208
12.2
Challenges for Extending Batch Duration and Productivity
210
12.2.1
Providing Energy
210
12.2.2
Stabilizing the Substrates
213
12.3
Scale-up of Reactions not Requiring Oxygen in Batch Mode
216
12.3.1
Test-tube Scale-up Results are Disappointing
216
Contents
XI
12.3.2
Thin-film
Format
Conserves
Performance 216
12.3.3
Investigating
Fundamental
Influences
218
\1
A Scale-up of Reactions Requiring Oxygen
218
12.4.1
Test-tube Scale up is Disastrous
218
12.4.2
Thin-film Format Conserves Performance
222
12.43
Stirred Tank Aerated Reactor Format Requires Antifoaming
Agents
222
12.4.4
Enhanced O2 Transfer Increases ATP Concentrations
226
12.4.5
Protein Production in 1-liter Batch Reactions
228
12.5
Conclusions and Projections
231
12.5.1
Personalized Medicine
231
12.5.2
Large-scale Pharmaceutical Production
232
Index
237 |
| adam_txt |
Contents
Preface XIII
List of Contributors
ХУЯ
1
Cell-free Protein Synthesis Systems: Historical Landmarks,
Classification, and General Methods
1
A. S. Spirin and]. R. Swartz
1.1
Introduction: Historical Landmarks
1
1.1.1
Discovery of Protein Synthesis in Cell Extracts
1
1.1.2
Translation of Exogenous Messages
1
1.1.3
Coupled Transcription-translation in Bacterial Extracts
2
1.1.4
Combined Transcription-translation Systems
3
1.1.5
Continuous Flow/Continuous Exchange Principle
3
1.2
Prokaryotic and Eukaryotic Types of Cell-free Expression
Systems
5
1.2.1
Cell Extracts
5
1.2.1.1
E. coli
extract
(ЕСЕ)
5
1.2.1.2
Wheat Germ Extract (WGE)
6
1.2.1.3
Rabbit Reticulocyte
Lysáte
(RRL)
6
1.2.2
Genetic Constructs (Expression Vectors)
7
1.2.2.1
Prokaryotic Systems
7
1.2.2.2
Eukaryotic Systems
8
1.3
Preparing Cell Extracts
11
1.3.1
E. coli
Extracts
11
1.3.1.1
Genetics
11
1.3.1.2
Cell Growth
13
1.3.1.3
Extract Preparation
14
1.3.2
Wheat Germ Extracts
15
1.4
Designing Reaction Composition
16
1.4.1
Mg2+ and Phosphate
16
1.4.2
Other Salts
18
1.4.3
Nucleotides and
Amino
Acids
18
1.4.4
Stabilization Reagents
ÍS
Cell-free Protein Synthesis. Edited by Alexander Spirin and James Swartz
Copyright
© 2008
WILEY-VCH
Verlag
GmbH
&
Co. KGaA,
Weinheim
ISBN:
978-3-527-31649-6
VI
I Contents
1.4.5
Other Factors
19
1.5
Providing Energy
19
1.5.1
Direct Nucleotide Regeneration
20
1.5.2
Indirect Nucleotide Regeneration
20
1.6
Enhancing Protein Folding
21
1.6.1
Temperature Effects
21
1.6.2
Cell Extract Concentration
23
1.6.3
Effects of Folding Ligands
23
1.6.4
Effects of Chaperones and Foldases
24
1.6.5
Effects of Detergents
24
2
The Constructive Approach for Cell-free Translation
35
T. Ueda
2.1
Introduction
35
2.2
The Process of Protein Synthesis
36
2.2.1
Polypeptide Synthesis
36
2.2.2
Protein Maturation
38
2.3
A Constructive Approach to Protein Synthesis
40
2.3.1
In Vitro
Reconstitution
of Polypeptide Synthesis
40
2.3.2
Protocol of Protein Synthesis using PURE System
41
2.3.3
Addition of Protein Folding Machinery to the PURE System
42
2.3.4
Integration of a Membrane Targeting System with the PURE
system
46
2.3.5
Protein Synthesis using the PURE System containing Molecular
Chaperones
48
2.4
Conclusion
49
3
Functional Cenomic Analysis using Sequential Cell-free Protein
Synthesis
51
K. A. Woodrow and]. R. Swartz
3.1
Introduction
5Î
3.1.1
The Post-genomic Era
51
3.1.2
Cell-free Protein Synthesis (CFPS) as a Functional Proteomic
Tool
52
3.2
Developing an enabling Technology for Sequential Expression
Analysis
54
3.2.1
Improving Linear Template Stability
55
3.2.2
Improving PCR Reactions for generating Genomic Linear
Templates
56
3.2.3
Optimizing Cofactor Concentrations for Enzyme Activation
58
3.3
Demonstrating Functional Genomic Analysis with CFPS
61
3.3.1
Isolation and Expression of Genomic Targets
62
Contents
VII
3.3.2
Effects of Sample Library on
ß-Lactamase
Expression and
Activity
62
3.4
Conclusions and Projections
64
4
Cell-free Technology for Rapid Production of Patient-specific Fusion
Protein Vaccines
69
A. R. Gotrke.,]. Yang, G.
Kanter,
R.
Levy and
J. R.
Swartz
4.1
Introduction
69
4.1.1
Lymphoma and Fusion Protein Vaccine Treatments
69
4.1.2
Comparing Cell-free and In Vivo Production Systems
70
4.2
Developing the Fusion Protein Construct and the Cell-free
Production Process
71
4.2.1
Fusion-protein Production in the Cell-free System
71
4.2.2
Oxidized Reaction Conditions and DsbC Increase Soluble Protein
Yield
71
4.2.3
GM-CSF is more Active at the N-terminus of the Fusion Protein
Vaccine
73
4.2.4
New Linker Improves Fusion Protein Stability
75
4.2.5
Expression and Purification Scale-up for Vaccine Protein
Production
77
4.3
Fusion Proteins Raise Protective Antibodies
78
4.3.1
Design of Vaccine Constructs and Mouse Studies
78
4.3.2
Fusion Protein Vaccination Protects against Aggressive Tumors
79
4.3.3
Antibody Generation is enhanced by Fusion Partners
79
4.4
Conclusions and Projections
80
5
Bacterial Cell-free System for Highly Efficient Protein Synthesis
83
T. Kigawa, T. Matsuda, T. Yabuki and S. Yokoyama
5.1
Overview
83
5.2
Introduction
83
5.3
Coupled Transcription-Translation System based on
E. coli
Extract
84
5.4 DNA
Template Construction
84
5.5
Preparation of Cell Extract from
E. coli
85
5.6
Batch-mode Cell-free Reaction
87
5.7
Dialysis-mode Cell-free Reaction
88
5.8
Template
DNA 91
5.9
Reaction Temperature
92
5.10
Surface Area of the Dialysis Membrane
93
5.11
Stable-isotope Labeling for NMR Spectroscopy
93
5.12
Selenomethionine Incorporation for
Х
-Ray Crystallography
94
5.13
Automation
95
5.14
Conclusion
95
VIII Contents
б
The Use of the
Escherichìa coli
Cell-free Protein Synthesis for
Structural Biology and Structural Proteomics
99
T. Kigawa, M. Inoue, M. Aoki, T. Matsuda, T. Yahuki, E. Seki,
T. Harada, S. Watanabe and S. Yokoyama
6.1
Overview
99
6.2
Introduction
100
6.3
High-throughput Expression by PCR-based Small-scale Cell-free
Protein Synthesis
100
6.4
Fully Automated Protein Production using Middle-scale Cell-free
Protein Synthesis
203
6.5
NMR Screening
104
6.6
Large-scale Protein Production for Structure Determination
105
6.7
Discussion
107
7
The Wheat Germ Cell-free Protein Synthesis System 111
T. Sawasaki and Y. Endo
7.1
Overview 111
7.2
Development of a Highly Efficient Eukaryotic Cell-free Protein
Synthesis System 111
7.2.1
Preparation of a Highly Active and Robust Extract from Wheat
Embryos
112
7.2.1.1
Protocol for the Preparation of Wheat Germ Extract
[12] 115
7.2.2
mRNA
5'
and
3'
UTRs which Enhance Translation
115
7.2.3
Split-primer PCR for Genome-wide Generation of DNAs for
Transcription
119
7.2.3.1
Protocol for "Split-primer" PCR
[13] 121
First PCR
122
7.2.4
Bilayer Translation Reaction Method
122
7.2.5
Transcription and Translation in One Tube
123
7.2.5.1
Protocol for One-tube Protein Synthesis Reaction
124
7.2.6
Reaction Methods for Large-scale Protein Production
125
7.3
Completion of Protocols for the Wheat Cell-free System
126
7.3.1
Performance of the Wheat Cell-free System
127
7.3.2
Robotic Automation of the Cell-free Protein Synthesis
132
7.4
Application to High-throughput Biochemical Annotation of Genetic
Information
132
7.4.1
Genome-wide Functional Analysis
132
7.4.2
Preparation of Protein for NMR Spectroscopy
134
7.5
Conclusion
136
Contents
IX
8
Cell-free
Expression
of
Integral Membrane Proteins
for Structural
Studies
141
С.
Klammt,
D.
Schwarz,
I.
Lehner,
S.
Sobhanifar,
F. Uhr,
J. Zeelen,
C. Glaubitz,
V.
Dötsch and F. Bernhard
8.1
Overview
141
8.2
Introduction
141
8.3
Specific Characteristics for the Cell-free Expression of Membrane
Proteins
143
8.3.1
Cell-free Expression of Membrane Proteins in the Presence of
Detergents or Lipids
145
8.3.2
Detergents for the Efficient Resolubilization of Cell-free Produced
Membrane Proteins
149
8.4
Case Studies for the High Level Cell-free Expression of Membrane
Proteins
150
8.4.1
a-Helical Transporters
150
8.4.2
G-Protein Coupled Receptors
252
8.4.3
^-Barrel Proteins
153
8.5
Structural Characterization of Cell-free Produced Membrane
Proteins
154
8.5.1
Crystallization of Cell-free Produced Membrane Proteins
154
8.5.2
Cell-free Expression as a Tool for High-resolution NMR
Spectroscopy
155
8.5.3
Applications of Cell-free Expression for Solid-state NMR
159
9
Cell-free Production of Membrane Proteins in the Presence of
Detergents
165
J.-M. Betton and M.
Miot
9.1
Introduction
165
9.2
Histidine Protein Kinases
166
9.3
Materials and Methods
168
9.3.1
Plasmids
368
9.3.2
Cell-free Protein Production
168
9.3.3
Protein Purification
168
9.3.4
Structural and Functional Protein Characterizations
169
9.4
Results and Discussion
169
9.4.1
Analytical Cell-free Production of
HÍS6
-tagged Proteins
169
9.4.2
Detergents Compatible with Cell-free Synthesis
171
9.4.3
Fidelity of In Vitro Biosynthesis Reactions in the Presence of
Brij35
173
9.4.4
High-level Production of Functional HPKs in CECF Technology
174
9.5
Conclusions
177
X I Contents
10
Novel Techniques using PCR and Cell-free Protein Synthesis Systems
for Combinatorial
Bioengineering 179
Η.
Nakano
and
T. Yamane
10.1
Introduction
379
10.2
Improvements in the
Escherìchia coli
Cell-free Protein Synthesis
Systems
180
10.3
High-throughput Construction of a Protein Library by SIMPLEX
180
10.3.1
Development of SIMPLEX
180
10.3.2
Quality of the SIMPLEX-based Protein Library
182
10.3.3
Expansion of the SIMPLEX-based Library
182
10.3.4
Application of SIMPLEX for Combinatorial Engineering of
Proteins
184
10.4
Development and Application of
SICREX
186
10.5
Conclusion
188
Π
Gene Cloning and Expression in Molecular Colonies
191
A. B.
Chetverìn,
T. R.
Samalov and
H. V. Chetverina
11.1
A Gap in Cell-free Biotechnology 1
91
11.2
Molecular Colony Technique
292
11.3
Gene Cloning in Molecular Colonies
193
11.4
Gene Expression in Molecular Colonies: Transcription
196
11.5
Gene Expression in Molecular Colonies: Translation
196
11.6
Gene Expression in Molecular Colonies: The Role of Thiol
Compounds
198
11.7
Conclusions
200
11.8
Molecular Colony Protocols
201
11.8.1
Amplification Gels
201
11.8.2
Growing
DNA
Colonies
202
11.8.3
Detection of Molecular Colonies
202
11.8.4
Transcription in Molecular Colonies
203
11.8.5
Protein Synthesis in Molecular Colonies
203
12
Large-Scale Batch Reactions for Cell-free Protein Synthesis
207
A. M. Voloshin and]. R. Swartz
12.1
Introduction
207
12.1.1
Cell-free Protein Synthesis
207
12.1.2
Comparing Cell-free Reaction Configurations; Advantages of Batch
Mode
208
12.2
Challenges for Extending Batch Duration and Productivity
210
12.2.1
Providing Energy
210
12.2.2
Stabilizing the Substrates
213
12.3
Scale-up of Reactions not Requiring Oxygen in Batch Mode
216
12.3.1
Test-tube Scale-up Results are Disappointing
216
Contents
XI
12.3.2
Thin-film
Format
Conserves
Performance 216
12.3.3
Investigating
Fundamental
Influences
218
\1
A Scale-up of Reactions Requiring Oxygen
218
12.4.1
Test-tube Scale up is Disastrous
218
12.4.2
Thin-film Format Conserves Performance
222
12.43
Stirred Tank Aerated Reactor Format Requires Antifoaming
Agents
222
12.4.4
Enhanced O2 Transfer Increases ATP Concentrations
226
12.4.5
Protein Production in 1-liter Batch Reactions
228
12.5
Conclusions and Projections
231
12.5.1
Personalized Medicine
231
12.5.2
Large-scale Pharmaceutical Production
232
Index
237 |
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| spelling | Cell-free protein synthesis methods and protocols ed. by Alexander S. Spirin ... Cell free protein synthesis Weinheim Wiley-VCH 2008 XX, 242 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Cell-Free System physiology Genetic translation Protein Biosynthesis Proteins Synthesis Translation Genetik (DE-588)4185909-1 gnd rswk-swf Proteinbiosynthese (DE-588)4175987-4 gnd rswk-swf Zellfreies System (DE-588)4342432-6 gnd rswk-swf (DE-588)4143413-4 Aufsatzsammlung gnd-content Zellfreies System (DE-588)4342432-6 s Proteinbiosynthese (DE-588)4175987-4 s Translation Genetik (DE-588)4185909-1 s DE-604 Spirin, Aleksandr S. 1931-2020 Sonstige (DE-588)124243266 oth text/html http://deposit.dnb.de/cgi-bin/dokserv?id=2878010&prov=M&dok_var=1&dok_ext=htm Inhaltstext Digitalisierung UB Regensburg application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=015437003&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Cell-free protein synthesis methods and protocols Cell-Free System physiology Genetic translation Protein Biosynthesis Proteins Synthesis Translation Genetik (DE-588)4185909-1 gnd Proteinbiosynthese (DE-588)4175987-4 gnd Zellfreies System (DE-588)4342432-6 gnd |
| subject_GND | (DE-588)4185909-1 (DE-588)4175987-4 (DE-588)4342432-6 (DE-588)4143413-4 |
| title | Cell-free protein synthesis methods and protocols |
| title_alt | Cell free protein synthesis |
| title_auth | Cell-free protein synthesis methods and protocols |
| title_exact_search | Cell-free protein synthesis methods and protocols |
| title_exact_search_txtP | Cell-free protein synthesis methods and protocols |
| title_full | Cell-free protein synthesis methods and protocols ed. by Alexander S. Spirin ... |
| title_fullStr | Cell-free protein synthesis methods and protocols ed. by Alexander S. Spirin ... |
| title_full_unstemmed | Cell-free protein synthesis methods and protocols ed. by Alexander S. Spirin ... |
| title_short | Cell-free protein synthesis |
| title_sort | cell free protein synthesis methods and protocols |
| title_sub | methods and protocols |
| topic | Cell-Free System physiology Genetic translation Protein Biosynthesis Proteins Synthesis Translation Genetik (DE-588)4185909-1 gnd Proteinbiosynthese (DE-588)4175987-4 gnd Zellfreies System (DE-588)4342432-6 gnd |
| topic_facet | Cell-Free System physiology Genetic translation Protein Biosynthesis Proteins Synthesis Translation Genetik Proteinbiosynthese Zellfreies System Aufsatzsammlung |
| url | http://deposit.dnb.de/cgi-bin/dokserv?id=2878010&prov=M&dok_var=1&dok_ext=htm http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=015437003&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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