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This detailed volume provides a toolbox for designing constructs, tackling expression and solubility issues, handling membrane proteins and protein complexes, and exploring innovative engineering of E. coli. The topics are largely grouped under four parts: high-throughput  cloning, expression screening, and optimization of expression conditions, protein production and solubility enhancement, case studies to produce challenging proteins and specific protein families, as well as applications of E. coli expression. Written for the highly successful Methods in Molecular Biology series, 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. 
Authoritative and practical, Heterologous Gene Expression in E. coli: Methods and Protocols serves molecular biologists, biochemists and structural biologists, those in the beginning of their research careers to those in their prime, to give both an historical and modern overview of the methods available to express their genes of interest in this exceptional organism.

Heterologous Gene Expression in E.coli: Methods and Protocols

Part I: High-Throughput Cloning, Expression Screening, and Optimization

 

1. Recombinant Protein Expression in E. coli: A Historical Perspective

            Opher Gileadi

 

2. N- and C-Terminal Truncations to Enhance Protein Solubility and Crystallization: Predicting Protein Domain Boundaries with Bioinformatics Tools

            Christopher D.O. Cooper and Brian D. Marsden

 

3. Harnessing the Profinity eXact^TM System for Expression and Purification of Heterologous Proteins in E. coli

            Yoav Peleg, Vadivel Prabahar, Dominika Bednarczyk, and Tamar Unger

 

4. ESPRIT: A Method for Defining Soluble Expression Constructs in Poorly Understood Gene Sequences

            Philippe J. Mas and Darren J. Hart

 

5. Optimizing Expression and Solubility of Proteins in E. coli Using Modified Media and Induction Parameters

            Troy Taylor, John-Paul Denson, and Dominic Esposito

 

6. Optimization of Membrane Protein Production Using Titratable Strains of E. coli

            Rosa Morra, Kate Young, David Casas-Mao, Neil Dixon, and Louise E. Bird

 

7. Optimizing E. coli-Based Membrane Protein Production Using Lemo21(DE3) or pReX and GFP-Fusions

            Grietje Kuipers, Markus Peschke, Nurzian Bernsel Ismail, Anna Hjelm, Susan Schlegel, David Vikström, Joen Luirink, and Jan-Willem de Gier

 

8. High Yield of Recombinant Protein in Shaken E. coli Cultures with Enzymatic Glucose Release Medium EnPresso B

            Kaisa Ukkonen, Antje Neubauer, Vinit J. Pereira, and Antti Vasala

 

Part II: Protein Purification and Solubility Enhancement

 

9. A Generic Protocol for Purifying Disulphide-Bonded Domains and Random Protein Fragments Using Fusion Proteins with SUMO3 and Cleavage by SenP2 Protease

            Hüseyin Besir

 

10. A Strategy for Production of Correctly Folded Disulfide-Rich Peptides in the Periplasm of E. coli

            Natalie J. Saez, Ben Cristofori-Armstrong, Raveendra Anangi, and Glenn F. King

 

11. Split GFP Complementation as Reporter of Membrane Protein Expression and Stability in E. coli: A Tool to Engineer Stability in a LAT Transporter

            Ekaitz Errasti-Murugarren, Arturo Rodríguez-Banqueri, and José Luis Vázquez-Ibar

 

12. Acting on Folding Effectors to Improve Recombinant Protein Yields and Functional Quality

            Ario de Marco

 

13. Protein Folding Using a Vortex Fluidic Device

            Joshua Britton, Joshua N. Smith, Colin L. Raston, and Gregory A. Weiss

 

14. Removal of Affinity Tags with TEV Protease

            Sreejith Raran-Kurussi, Scott Cherry, Di Zhang, and David S. Waugh

 

Part III: Case Studies to Produce Challenging Proteins and Specific Protein Families

 

15. Generation of Recombinant N-Linked Glycoproteins in E. coli

            Benjamin Strutton, Stephen R.P. Jaffé, Jagroop Pandhal, and Phillip C. Wright

 

16. Production of Protein Kinases in E. coli

            Charlotte A. Dodson

 

17. Expression of Prokaryotic Integral Membrane Proteins in E. coli

            James D. Love

 

18. Multiprotein Complex Production in E. coli: The SecYEG-SecDFYajC-YidC Holotranslocon

            Imre Berger, Quiyang Jiang, Ryan J. Schulze, Ian Collinson, and Christiane Schaffitzel

 

19. Membrane Protein Production in E. coli Lysates in Presence of Pre-Assembled Nanodiscs

            Ralf-Bernhardt Rues, Alexander Gräwe, Erik Henrich, and Frank Bernhard

 

20. Not Limited to E. coli: Versatile Expression Vectors for Mammalian Protein Expression

            Katharina Karste, Maren Bleckmann, and Joop van den Heuvel

 

21. A Generic Protocol for Intracellular Expression of Recombinant Proteins in Bacillus subtilis

            Trang Phan, Huynh Ph ng, Tuom Truong, and Hoang Nguyen

 

Part IV: Applications of E. coli Expression

 

22. In Vivo Biotinylation of Antigens in E. coli

            Susanne Gräslund, Pavel Savitsky, and Susanne Müller-Knapp

 

23. Cold-Shock Expression System in E. coli for Protein NMR Studies

            Toshihiko Sugiki, Toshimichi Fujiwara, and Chojiro Kojima

 

24. High-Throughput Production of Proteins in E. coli for Structural Studies

            Charikleia Black, John J. Barker, Richard B. Hitchman, Hok Sau Kwong, Sam Festenstein, and Thomas B. Acton

 

25. Mass Spectrometric Analysis of Proteins

            Rod Chalk

 

26. How to Determine Interdependencies of Glucose and Lactose Uptake Rates for Heterologous Protein Production with E. coli

            David J. Wurm, Christoph Herwig, and Oliver Spadiut

 

27. Interfacing Biocompatible Reactions with Engineered Escherichia coli

            Stephen Wallace and Emily P. Balskus