Gene Expression Systems (eBook)

Front Cover 1
Gene Expression Systems: Using Nature for the Art of Expression 4
Copyright Page 5
Contents 6
Contributors 12
Introduction 14
The Art of Expression 14
Purpose of This Book 15
Selecting a Suitable Expression System: Considerations 16
Genomics and the Future of Protein Expression Systems 18
SECTION I: PROKARYOTIC EXPRESSION SYSTEMS 20
Chapter 1. Gene Expression Systems Based on Bacteriophage T7 RNA Polymerase 22
Introduction 23
T7 RNAP in Its Natural Habitat 24
Biochemistry of T7 RNAP 27
T7-Based Gene Expression Systems 37
Conclusions 49
References 50
Chapter 2. Expression Vectors Employing the trc Promoter 58
Introduction 59
Vectors for trc-Driven Expression 64
Additional Hosts for trc-Driven Expression 70
Outlook for Further Improvements of Expression Vectors Employing the trc Promoter 71
References 72
Chapter 3. Bacillus Expression" A Gram-Positive Model 78
Introduction 79
Bacilli Used as Industrial Production Organisms 80
Genetic Manipulation in Bacilli 84
Plasmid Vectors 88
Transcriptional Regulation 90
Conclusions 94
Appendix 95
References 102
Chapter 4. araB Expression System in Escherichia coli 108
Introduction 108
The araB Promoter and How It Works 110
Use of the ara System for Expression of Recombinant Products 112
Conclusions 117
References 119
SECTION II: EUKARYOTIC EXPRESSION SYSTEMS 122
Chapter 5. Adenoviral Vectors for Protein Expression 124
Adenovirus Biology 125
Adaptation of Adenovirus as a Gene Transfer Vector 130
Construction of Adenovirus Expression Vectors 132
Applications of Adenoviral Vectors in Gene Expression 141
Considerations in the Use of Adenovirus Expression Vectors 151
Advantages and Disadvantages of Adenoviral Vectors 154
Future Directions 156
Conclusions 157
References 158
Chapter 6. Expression in the Methylotrophic Yeast Pichia pastoris 170
Introduction 171
Background Information 174
Construction of Expression Strains 179
Important Considerations in the Use of the Expression System 188
Conclusion 196
References 197
Chapter 7. Recombinant Protein Expression in Pichia methanolica 206
Background 206
DNA Transformation 207
Expression Vector and Identification of an Expression Strain 209
Gene Disruptions and Generation of a Protease-Deficient Strain 211
Fermentation 212
Evaluation of Protein Expression 212
Expression of the 65-kDa Isoform of Human Glutamate 215
Protein Secretion 215
Benefits and Liabilities of the P. methanolica Expression System 218
Summary 219
Appendix 220
References 220
Chapter 8. Cytomegalovirus Promoter for Expression in Mammalian Cells 224
Introduction 225
The Cytomegalovirus Enhancer-Containing Promoter 227
Positive Regulation 229
Experimental Procedures 232
Effects Upstream of the Cytomegalovirus Enhancer 234
Effects Downstream of the Cytomegalovirus Promoter 235
Negative Regulation 236
Conclusions 238
References 241
Chapter 9. Inducible Mammalian Expression Systems 248
Background: Development of Inducible Expression Systems 248
Literature Review 258
Author's Experience 261
Comparing Inducible Mammalian Expression Systems Currently Available 263
Conclusions and Future Directions 263
References 266
Chapter 10. Protein Expression in Mammalian Cells Using Sindbis Virus 272
Introduction 273
Applications 280
Protein Expression 285
Comparisons with Other Systems 292
New Directions and Conclusions 295
References 296
SECTION III: EXPRESSION IN INSECT SYSTEMS 300
Chapter 11. Drosophila S2 System for Heterologous Gene Expression 302
Introduction 303
Properties of S2 Line 303
Experimental Procedures 305
Literature Review 313
Choosing an Expression System 328
Future Directions 331
Conclusions 334
Appendix 334
References 338
Chapter 12. Baculovirus Expression Vector System 344
Introduction 345
Background 347
Basic Research of Baculoviruses as a Tool for Gene Expression 360
Expression Examples 362
Guidelines to Optimize Heterologous Gene Expression in Baculovirus 365
Summary and Future 367
Appendix 368
References 372
SECTION IV: TRANSGENIC EXPRESSION 378
Chapter 13. Recombinant Protein Expression in Transgenic Mice 380
Overview and Background 381
Transgenic Mice: General Overview 381
Examples of Recombinant Protein Expression in Transgenic Mice 384
Modeling Human Disease through Targeted Overexpression 391
Biomass: Transgenic Mice as Model Bioreactors 394
Future of Transgenic Mice Approaches: Need for Inducible Expression 396
Conclusions 403
References 404
Chapter 14. Expression of Recombinant Proteins in the Milk of Transgenic Animals 412
Introduction 413
Expression of Heterologous Proteins in Milk 414
Milk-Specific Transgenes 416
Insertion of the Transgene into the Germ Line 418
Transgenic Animal Production 421
Biosynthesis of Milk Proteins 422
Milk Secretion from the Mammary Gland 425
Lactation and Milk Output 425
Milk Composition and Purification of the Target Protein 426
Quality Issues in Transgenic Production 430
Regulatory Considerations 431
Current Status and Future Directions 433
References 434
Chapter 15. Recombinant Protein Expression in Plants 442
General Introduction 443
Transformation Methods 444
Promoters Used for Recombinant Protein Accumulation in Plants 448
Expression of Recombinant Proteins in Plants and Agricultural Biotechnology 451
Recombinant Protein Expression in Plants to Obtain New Products 455
Protein Targeting and Accumulation 460
Virus-Mediated Expression Systems 461
Summary/Discussion 463
References 465
Index 476