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With insolubility proving to be one of the most crippling bottlenecks in the protein production and purification process, this volume serves to aid researchers working in the recombinant protein production field by describing a wide number of protocols and examples. Insoluble Proteins: Methods and Protocols includes chapters that describe not only the recombinant protein production in different expression systems but also different purification and characterization methods to finally obtain these difficult-to-obtain proteins. Beginning with protein production methods using both prokaryotic and eukaryotic expression systems, the book continues with purification protocols using insoluble proteins, the characterization of insoluble proteins, as well as a general overview of interesting applications of insoluble proteins. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Comprehensive and practical, Insoluble Proteins: Methods and Protocols aims to provide the scientific community with detailed and reliable state-of-the-art protocols that are used in order to successfully produce and purify recombinant proteins prone to aggregate.

Insoluble Proteins: Methods and Protocols

1. General Introduction: Recombinant Protein Production and Purification of Insoluble Proteins

Neus Ferrer-Miralles, Paolo Saccardo, José Luis Corchero, Zhikun Xu, and Elena García-Fruitós

Part I: Recombinant Protein Production in Escherichia coli

2. Overcoming the Solubility Problem in E. coli: Available Approaches for Recombinant Protein Production

Agustín Correa and Pablo Oppezzo

3. Optimization of Culture Parameters and Novel Strategies to Improve Protein Solubility

Ranjana Arya, Jamal S.M. Sabir, Roop S. Bora, and Kulvinder S. Saini

4. Cleavable Self-Aggregating Tags (cSAT) for Protein Expression and Purification

Zhanglin Lin, Qing Zhao, Bihong Zhou, Lei Xing, and Wanghui Xu

5. Beyond the Cytoplasm of Escherichia coli: Localizing Recombinant Proteins Where You Want Them

Jason T. Boock, Dujduan Waraho-Zhmayev, Dario Mizrachi, and Matthew P. DeLisa

6. Characterization of Amyloid-Like Properties in Bacterial Intracellular Aggregates

Anna Villar-Pique, Susanna Navarro, and Salvador Ventura

Part II: Strategies to Produce Insoluble Proteins in Cell-Free Expression Systems

7. Co-Translational Stabilization of Insoluble Proteins in Cell-Free Expression Systems

Lei Kai, Erika Orbán, Erik Henrich, Davide Proverbio, Volker Dötsch, and Frank Bernhard

Part III: Recombinant Protein Production in Lactic Acid Bacteria (LAB)

8. Functional Expression of Plant Membrane Proteins in Lactococcus lactis

Sylvain Boutigny, Emeline Sautron, Annie Frelet-Barrand, Lucas Moyet, Daniel Salvi, Norbert Rolland, and Daphné Seigneurin-Berny

Part IV: Recombinant Protein Production in Yeast

9. High Cell-Density Expression System: Yeast Cells in a Phalanx Efficiently Produce a Certain Range of "Difficult-to-Express" Secretory Recombinant Proteins

Yasuaki Kawarasaki, Takeshi Kurose, and Keisuke Ito

Part V: Recombinant Protein Production in Insect Cells-Baculovirus

10. Insect Cells-Baculovirus System for the Production of Difficult to Express Proteins

Judit Osz-Papai, Laura Radu, Wassim Abdulrahman, Isabelle Kolb-Cheynel, Nathalie Troffer-Charlier, Catherine Birck, and Arnaud Poterszman

Part VI: Recombinant Protein Production in Mammalian Cells

11. Transient Expression in HEK 293 Cells: An Alternative to E. coli for the Production of Secreted and Intracellular Mammalian Proteins

Joanne E. Nettleship, Peter J. Watson, Nahid Rahman-Huq, Louise Fairall, Mareike G. Posner, Abhishek Upadhyay, Yamini Reddivari, Jonathan M.G. Chamberlain, Simon E. Kolstoe, Stefan Bagby, John W.R. Schwabe, and Raymond J. Owens

12. Recombinant Glycoprotein Production in Human Cell Lines

Kamilla Swiech, Marcela Cristina Correa de Freitas, Dimas Tadeu Covas, and Virgínia Picanço-Castro

Part VII: Recombinant Protein Production in Other Systems

13. Soluble Recombinant Protein Production in Pseudoaltermonas haloplanktis TAC125

Maria Giuliani, Ermenegilda Parrilli, Filomena Sannino, Gennaro Apuzzo, Gennaro Marino, and Maria Luisa Tutino

Part VIII: Insoluble Protein Purification

14. A Screening Methodology for Purifying Proteins with Aggregation Problems

Mario Lebendiker, Michal Maes, and Assaf Friedler

15. Solubilization and Refolding of Inclusion Body Proteins

Anupam Singh, Vaibhav Upadhyay, and Amulya K. Panda

16. Bacterial Inclusion Body Purification

Joaquin Seras-Franzoso, Spela Peternel, Olivia Cano-Garrido, Antonio Villaverde, and Elena García-Fruitós

17. Characterization of Intracellular Aggresomes by Fluorescent Microscopy

Lianwu Fu and Elizabeth Sztul

Part IX: Insoluble Protein Characterization

18. Dialysis: A Characterization Method of Aggregation Tendency

Mireia Pesarrodona, Ugutz Unzueta, and Esther Vázquez

19. Applications of Mass Spectrometry to the Study of Protein Aggregation

Sílvia Bronsoms and Sebastián A. Trejo

20. Insoluble Protein Assemblies Characterized by Fourier Transform Infrared Spectroscopy

Antonino Natalello and Silvia M. Doglia

21. Insoluble Protein Characterization by Circular Dichroism (CD) Spectroscopy and Nuclear Magnetic Resonance (NMR)

Shaveta Goyal, Haina Qin, Liangzhong Lim, and Jianxing Song

22. Methods for Characterization of Protein Aggregates

Witold Tatkiewicz, Elisa Elizondo, Evelyn Moreno, Cesar Díez-Gil, Nora Ventosa, Jaume Veciana, and Imma Ratera

23. Predicting the Solubility of Recombinant Proteins in Escherichia coli

Roger G. Harrison and Miguel J. Bagajewicz

Part X: Insoluble Protein Applications

24. Insoluble Protein Applications: The Use of Bacterial Inclusion Bodies as Biocatalysts

Eva Hrabárová, Lucia Achbergerová, and Jozef Nahálka