Heterologous Gene Expression in E.coli

1. Front Matter

   Pages i-xiv

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2. High-Throughput Cloning, Expression Screening, and Optimization

   1. Front Matter

      Pages 1-1

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

      • Opher Gileadi

      Pages 3-10

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

      • Christopher D. O. Cooper, Brian D. Marsden

      Pages 11-31

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

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

      Pages 33-43

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

      • Philippe J. Mas, Darren J. Hart

      Pages 45-63

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

      • Troy Taylor, John-Paul Denson, Dominic Esposito

      Pages 65-82

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

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

      Pages 83-107

   8. 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 et al.

      Pages 109-126

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

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

      Pages 127-137

3. Protein Purification and Solubility Enhancement

   1. Front Matter

      Pages 139-139

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

      • Hüseyin Besir

      Pages 141-154

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

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

      Pages 155-180

   4. 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, José Luis Vázquez-Ibar

      Pages 181-195

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

      • Ario de Marco

      Pages 197-210

   6. Protein Folding Using a Vortex Fluidic Device

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

      Pages 211-220

   7. Removal of Affinity Tags with TEV Protease

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

      Pages 221-230

4. Case Studies to Produce Challenging Proteins and Specific Protein Families

   1. Front Matter

      Pages 231-231

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

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

      Pages 233-250

   3. Production of Protein Kinases in E. coli

      • Charlotte A. Dodson

      Pages 251-264

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.

• Protein purification
• Recombinant proteins
• Escherichia coli
• Structural biology
• High-throughput cloning
• Expression screening
• Solubility enhancement

• SGC, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom

  Nicola A. Burgess-Brown

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