Physiological Stress Responses In Bioprocesses:9783540203117:Unknown:eCampus.com

Molecular Components of Physiological Stress Responses in Escherichia coli

L.M. Wick, T. Egli

(46)

1 Introduction

(1)

2 Molecular Components Involved in Stress Response Regulation

(3)

2.1 Nucleic Acids

(1)

2.1.1 DNA

(1)

2.1.2 RNA

(1)

2.2 Proteins

(2)

2.3 Small Molecular Weight Effectors

(1)

3 The Heat Shock Response

(8)

3.1 Regulation of the Heat Shock Response 8

3.1.1 Transcriptional Regulation 9

3.1.2 Translational Regulation 11

3.1.3 Posttranslational Regulation 11

3.2 Protein Folding and Degradation Control

(3)

3.2.1 Chaperones

(2)

3.2.2 Proteases

(1)

3.2.3 Posttranslational Quality Control

(1)

4 The Envelope Stress Response

(4)

4.1 The σE Response

(1)

4.2 The Cpx Response

(2)

4.3 The Bae Response

(1)

5 The Cold Shock Response

(7)

5.1 Cold Shock Induced Proteins

(2)

5.2 CspA - The Major Cold Shock Protein

(2)

5.3 The CspA Family

(1)

5.4 Sensing of Cold Shock

(1)

5.5 Changes in Membrane Composition

(1)

6 The Stringent Response

(6)

6.1 Regulation of (p)ppGpp Synthesis and Decay

(4)

6.1.1 RelA

(1)

6.1.2 SpoT

(2)

6.2 Effects and Mechanisms of (p)ppGpp

(2)

6.2.1 Effects of (p)ppGpp

(1)

6.2.2 Mechanisms of (p)ppGpp Regulation

(1)

6.2.3 Growth Rate Control by (p)ppGpp

(1)

7 The General Stress Response

(5)

7.1 Regulation of σS

(3)

7.1.1 Transcriptional Regulation

(2)

7.1.2 Translational Regulation

(1)

7.1.3 Posttranslational Regulation

(1)

7.2 Effects of σS

(14)

7.2.1 Physiological Effects of σS

(1)

7.2.2 σS-Dependent Promoters

(1)

7.2.3 Role of σS in Various Habitats

(1)

8 Conclusions and Perspectives

(1)

9 References

(9)

Monitoring of Stress Responses

T. Schweder M. Hecker

(26)

1 Introduction

(1)

2 RNA Analysis Techniques

(7)

2.1 Classical Techniques for the Analysis of mRNA Levels

(1)

2.2 Expression Analysis by Optical DNA-Chips

(2)

2.3 Alternative RNA Analysis Techniques

(4)

3 Stress Responses of Industrially Relevant Microorganisms

(3)

4 Monitoring of Bioprocess Relevant Stress

(6)

4.1 The Scale-Up of Microbial Bioprocesses

(2)

4.2 The Cellular Responses to the Overproduction of Recombinant Proteins

(3)

5 Strain Design

(3)

6 Outlook

(1)

7 References

(5)

Stress Induced by Recombinant Protein Production in Escherichia coli

F. Hoffmann, U. Rinas

(20)

1 Introduction

(1)

2 Metabolic Consequences of Recombinant Protein Production

(4)

2.1 Inhibition of Growth

(1)

2.2 Modification of Catabolism

(2)

2.2.1 Metabolic Burden and Stress Load

(1)

2.2.2 Catabolic Flux Adjustment

(1)

2.2.3 Adjustment of the Energy Generating Enzyme System

(1)

2.3 Modification of Anabolism

(2)

2.3.1 Anabolic Flux Adjustment

(1)

2.3.2 Adjustment of the Protein Producing System

(1)

3 DNA Replication

(2)

3.1 Chromosomal DNA

(1)

3.2 Replication of Plasmid DNA

(1)

4 Induction of Stress Responses

(5)

4.1 Heat-Shock Response

(1)

4.2 Stringent Response

(1)

4.3 SOS Response

(1)

4.4 Overlapping Stress Responses

(1)

4.5 On-line Techniques for Stress Monitoring

(1)

5 Conclusions

(3)

5.1 Is There a Limited Adaptation Capacity?

(1)

5.2 Can Stress Be Reduced by Gradual Induction?

(1)

5.3 Should Stress Be Minimized for Optimum Protein Production?

(1)

6 References

(4)

Inclusion Bodies: Formation and Utilisation

B. Fahnert, H. Lilie, P. Neubauer

(50)

1 Introduction

(1)

2 Protein Aggregation in Prokaryotes - The Formation of IBs

(26)

2.1 Structural Characteristics of Proteins Favouring Aggregation

(3)

2.1.1 Disulfide Bonds

(1)

2.1.2 Membrane Proteins

(1)

2.1.3 Glycosylation

(1)

2.2 Composition and Structure of IBs and Kinetics of IB Formation

(4)

2.2.1 Architecture and Structure

(1)

2.2.2 Composition of IBs

100

(1)

2.2.3 Kinetics of In Vivo Aggregation

101

(2)

2.2.4 Stability of IBs

103

(1)

2.3 The Physiology of IB Formation

103

(11)

2.3.1 The Metabolic Load of IB Synthesis

103

(4)

2.3.2 The Response to Misfolded Protein

107

(1)

2.3.2.1 Stress Responses

107

(1)

2.3.2.2 Chaperone Action

110

(1)

2.3.2.3 Periplasmic Response to Misfolded Protein

112

(1)

2.3.2.4 Response to Misfolded Proteins in Other Organisms

112

(1)

2.3.3 Host Characteristics for High-Quality IBs

113

(1)

2.4 IB Based Processes Versus Soluble Production

114

(7)

2.4.1 Cultivation Conditions Promoting Aggregation

114

(1)

2.4.2 IBs as a Result of Failure in Formation of Correct Disulfide Bonds

114

(1)

2.4.3 How to Avoid IBs and to Favour Correctly Folded Proteins

115

(1)

2.4.3.1 Rate of Synthesis

115

(1)

2.4.3.2 Fusion Proteins

116

(1)

2.4.3.3 Coexpression of Chaperones and Foldases

117

(1)

2.4.3.4 Cultivation Conditions and Addition of Folding Promoting Agents

119

(1)

2.4.3.5 Cellular Redox Situation

121

(1)

2.5 IBs in Prokaryotes Other than E. Coli

121

(1)

3 Production of IBs and Down-Stream Functionalisation

122

(14)

3.1 Fermentation Process for IB Protein Production

122

(5)

3.2 Preparation of IBs

127

(2)

3.2.1 IB Isolation

127

(1)

3.2.2 Purification of IBs

128

(1)

3.2.3 Solubilisation of IBs

128

(1)

3.3 Refolding of Proteins from IBs

129

(5)

3.3.1 Disulfide Bond Formation During Protein Renaturation

131

(1)

3.3.2 Improving Renaturation

132

(2)

3.4 Industrial Processes Based on Refolding of IB Proteins

134

(1)

3.4.1 Human Tissue-Type Plasminogen Activator (t-PA)

134

(1)

3.4.2 Antibody Fragments and Immunotoxins

135

(1)

3.5 The Future of IB Based Processes for Recombinant Proteins

135

(1)

4 References

136

(7)

Roles of Heat-Shock Chaperones in the Production of Recombinant Proteins in Escherichia coli

F. Hoffmann, U. Rinas

143

(20)

1 Introduction

144

(1)

2 Recombinant Protein Production at Modified Concentrations of Heat-Shock Chaperones

145

(2)

2.1 Effects of Chaperone Gene Overexpression or Elimination

145

(1)

2.2 Choice of Chaperone Systems

146

(1)

3 Substrate Specificities and Functions of Chaperones

147

(9)

3.1 Hsp70 System: DnaK, DnaJ, and GrpE

147

(4)

3.1.1 Structure and Function

147

(3)

3.1.2 Role of DnaK in the Chaperone Network

150

(1)

3.1.3 Role of DnaK in Regulation of the Heat-Shock Response

151

(1)

3.2 Hsp60 System: GroEL and GroES

151

(1)

3.3 Small Heat-Shock Proteins (sHsps): IbpA and IbpB

152

(2)

3.3.1 Structure and Function

152

(1)

3.3.2 Homologous sHsps in Other Organisms

153

(1)

3.4 Hsp100 System: The Clp Family

154

(11)

3.4.1 Clp Proteases

154

(1)

3.4.2 ClpB

155

(1)

4 Outlook

156

(2)

5 References

158

(5)

Analysis and Control of Proteolysis of Recombinant Proteins in Escherichia coli

A. Rozkov, S.-O. Enfors

163

(34)

1 Role of Proteolysis

164

(1)

2 E. coli Proteases

165

(4)

2.1 Lon (La) Protease

166

(1)

2.2 C1pAP (Ti) Protease

167

(1)

2.3 C1pYQ (Hs1UV) Protease

167

(1)

2.4 Proteases of the Cell Envelope

168

(1)

3 Energy-Dependence

169

(1)

4 Susceptibility to Proteolysis

169

(1)

5 Impact of Proteolysis on the Yield of Recombinant Proteins

170

(1)

6 Measurements of Proteolysis

171

(1)

7 Strategies to Control Proteolysis in E. coli

172

(13)

7.1 Control of Proteolysis on the Protein Level

173

(1)

7.1.1 Sequence Modification

173

(1)

7.1.2 Protective Fusion

173

(1)

7.1.3 Inclusion Body Formation Control

173

(1)

7.2 Control of Proteolysis on Cell Level

174

(2)

7.2.1 Use of Protease Mutations

174

(1)

7.2.2 Use of Host Strain Deficient in the Stringent Response

175

(1)

7.2.3 Co-Expression of Protease Inhibitors

175

(1)

7.2.4 Secretion to Periplasm

176

(1)

7.3 Control of Proteolysis on Cultivation Level

176

(8)

7.3.1 Temperature Optimisation

176

(1)

7.3.2 Optimisation of pH

177

(1)

7.3.3 Addition of Protease Inhibitors to the Culture Medium

177

(1)

7.3.4 Use of Complete Medium or Supplementation of Amino Acids

177

(1)

7.3.5 Effects of Starvation and Extreme Growth Limitation in High-Cell-Density Fed-Batch Cultures

178

(2)

7.3.6 Influence of Toxic Metabolic Products

180

(1)

7.3.7 Optimisation of Induction Strategy

181

(1)

7.3.8 Control of Scale-Up-Specific Effects

182

(2)

7.4 Downstream Processing Level

184

(1)

8 Concluding Remarks

185

(1)

9 References

186

(11)

The Application of Multi-Parameter Flow Cytometry to Monitor Individual Microbial Cell Physiological State

C.J. Hewitt, G. Nebe-Von-Caron

197

(28)

1 Introduction

198

(1)

2 Classical Microbiological Techniques

198

(2)

2.1 Bulk Measurements

198

(1)

2.2 Single Cell Measurements

199

(1)

3 Flow Cytometry

200

(12)

3.1 Introduction

200

(1)

3.2 Practical Considerations

201

(1)

3.3 Theoretical Considerations

202

(10)

3.3.1 Light Scatter

202

(4)

3.3.2 Physiological Studies

206

(6)

4 Practical Applications of Multi-Parameter Flow Cytometry

212

(8)

4.1 Studies on High Cell Density Fed-Batch Bacterial Fermentations

212

(7)

4.1.1 Escherichia coli

213

(5)

4.1.2 Rhodococcus Sp

218

(1)

4.2 Bioremediation of Heavy Metal Contaminated Waste Waters

219

(1)

4.3 Studies on Substrate Toxicity in the Indene Biotransformation

219

(1)

5 Conclusions

220

(1)

6 References

221

(4)

Author Index Volumes 51-89

225

(16)

Subject Index

241


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