Cell–free Protein Synthesis: Methods and Protocols

Complex biomolecules such as proteins cannot simply be synthesized by standard chemical reactions. They need to be assembled making use of the biological mechanisms that generate them in the living cell. Recreating the biosynthesis of proteins in a controlled cell–free laboratory system is no trivial task, yet brings enormous benefits in terms of product cost and purity that are particularly relevant for biotechnological applications.
With its detailed description of membrane protein expression, high–throughput and genomic–scale expression studies, this practical handbook covers the latest advances in the field –– both on the analytical and the preparative scale.
Following an introduction, the book goes on to deal with the constructive approach for cell–free translation, recombinant human proteins, and cell–free expression and production. It also covers novel techniques using PCR and cell–free protein synthesis systems for combinatorial bioengineering, as well as gene cloning and expression in molecular colonies.
The step–by–step protocols and practical examples given for each method represent practical advice for beginners and experts alike.

Spis treści:
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 Prot ein 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 CellR 11;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.

Nota biograficzna:
Alexander Spirin is a Professor at the Moscow State University, and the director of the Protein Research Institute of the Russian Academy of Sciences in Pushchino, Russia. He is one of the pioneers in the biochemical analysis of protein synthesis and has worked in the field for more than 40 years, performing the first–ever in vitro synthesis of proteins by using ribosome components. Professor Spirin has received many honors for his scientific achievements, including the Hans Adolf Krebs Medal, and is the author of several books on ribosomes and protein synthesis.
James Swartz is Professor of Chemical Engineering at Stanford University, California, USA. A graduate of the Massachusetts Institute of Technology, he has pioneered protein synthesis on an industrial scale while working for the companies Eli Lilly and Genentech. He is a leading expert on the biotechnological synthesis of mammalian proteins using bacterial whole cells and cell extracts.

Okładka tylna:
Complex biomolecules such as proteins cannot simply be synthesized by standard chemical reactions. They need to be assembled making use of the biological mechanisms that generate them in the living cell. Recreating the biosynthesis of proteins in a controlled cell–free laboratory system is no trivial task, yet brings enormous benefits in terms of product cost and purity that are particularly relevant for biotechnological applications.
With its detailed description of membrane protein expression, high–