Preface.
List of Contributors.
1. Cell-free Protein Synthesis Systems: Historical Landmarks, Classification, and General Methods (A. S. Spirin and J. R. Swartz).
1.1. Introduction: Historical Landmarks.
1.2. Prokaryotic and Eukaryotic Types of Cell-free Expression Systems.
1.3. Preparing Cell Extracts.
1.4. Designing Reaction Composition.
1.5. Providing energy.
1.6. Enhancing Protein Folding.
2. The Constructive Approach for Cell-free Translation (T. Ueda).
2.1. Introduction.
2.2. The Process of Protein Synthesis.
2.3. A Constructive Approach to Protein Synthesis.
2.4. Conclusion.
3. Functional Genomics Analysis using Sequential Cell-free Protein Synthesis (K. A. Woodrow and J. R. Swartz).
3.1. Introduction.
3.2. Developing an enabling Technology for Sequential expression Analysis.
3.3. Demonstrating Functional Genomic Analysis with CFPS.
3.4. Conclusions and Projections.
4. Cell-free Technology for Rapid Production of Patient-specified Fusion Protein Vaccines (A. R. Goerke J. Yang, G. Kanter, R. Levy and J. R. Swartz).
4.1. Introduction.
4.2. Developing the Fusion Protein Construct and the Cell-Free Production Process.
4.3. Fusion Proteins Raise Protective Antibodies.
4.4. Conclusions and Projections.
5. Bacterial Cell-free System for Highly Efficient Protein Synthesis (T. Kigawa, T. Matsuda, T. Yabuki and S. Yokoyama).
5.1. Overview.
5.2. Introduction.
5.3. Coupled Transcription-Translation System abased on E. coli Extract.
5.4. DNA Template Construction.’
5.5. Preparation of Cell Extract from E. Coli.
5.6. Batch-mode Cell-free Reaction.
5..7. Dialysis-mode Cell-free Reaction.
5.8. Te4mplate DNA.
5.9. Reaction Temperature.
5.10. Surface Area of the Dialysis Membrane.
5.11. Stable-isotope Labeling for NMR Spectroscopy.
5.12. Selenomethionine Incorporation for X-Ray Crystallography.
5.13. Automation.
5.14. Conclusion.
6. The Use of the Escherichia coli Cell-free Protein Synthesis for Structural Biology and Structural Proteomics (T. Kigawa, M. Inoue, M. Aoki, T. Matsuda, T. Yabuki, E. Seki, T. Harada, S. Watanabe and S. Yokoyama).
6.1. Overview.
6.2. Introduction.
6.3. High-throughput Expression by PCR-based Small-scale cell-free Protein Synthesis.
6.4. Fully Automated Protein Production using Middle-scale Cell-Free Protein Synthesis.
6.5. NMR Screening.
6..6. Large-scale Protein Production for Structure Determination.
6.7. Discussion.
7. The Wheat germ Cell-free Protein Synthesis System (T. Sawasaki and Y. Endo).
7.1. Overview.
7.2. Development of a Highly Efficient Eukaryotic Cell-free Protein Synthesis System.
7.3. Completion of Protocols for the Wheat cell-free System.
7.4. Application to High-throughput Biochemical Annotat5ion of Genetic Information.
7.5. Conclusion.
8. Cell-free Expression of Integral membrane Proteins for Structural Studies (C. Klammt, D. Schwarz, I. Lehner, S. Sobhanifar, F. Lohr, J. Zeelen, C. Glaubitz, V. Dotsch and F. Bernhard).
8.1. Overview.
8.2. Introduction.
8.3. Specific Characteristics for the Cell-free Expression of Membrane Proteins.
8.4. Case Studies for the High Level cell-free Expression of Membrane Proteins.
8.5. Structural Characterization of Cell-free Produced Membrane Proteins.
9. Cell-free Production of Membrane Proteins in the Presence of Detergents (J .-M. Betton and M. Miot).
9.1. Introduction.
9.2. Histidine Protein Kinases.
9.3. Materials and Methods.
9.4. Results and Discussion.
9.5. Conclusions.
10. Novel Techniques using PCR and Cell-free Protein Synthesis Systems for Combinatorial Bioengineering (H. Nakano and T. Yamane).
10.1. Introduction.
10.2. Improvements in the Escherichia coli Cell-free Protein Synthesis Systems.
10.3. High-throughput Construction of a Protein Library by SIMPLEX.
10.4. Development and Application of SICREX.
10.5. Conclusion.
11. Gene Cloning and expression in Molecular Colonies (A. B. Chetverin, T. R. Samatov and H. V. Chetverina).
11.1. A Gap in Cell-free Biotechnology.
11.2. Molecular Colony Technique.
11.3. Gene Cloning in Molecular Colonies.
11.4. Gene Expression in Molecular Colonies.
11.5. Gene Expression in Molecular Colonies: Transcription.
11.6. Gene Expression in Molecular colonies: The Role of Thiol Compounds.
11.7. Conclusions.
11.8. Molecular Colony Protocols.
12. Large-Scale Batch Reactions for Cell-free Protein Synthesis (A. M. Voloshin and J. R. Swartz).
12.1. Introduction.
12.2. Challenges for Extending Batch Duration and Productivity.
12.3. Scale-up of Reactions not requiring Oxygen in Batch Mode.
12.4. Scale-up of Reactions Requiring Oxygen.
12.5. Conclusions sand Projections.
Index.