Burgess-Brown Heterologous Gene Expression in E.coli

Methods and Protocols
1. Auflage 2017
ISBN: 978-1-4939-6887-9
Verlag: Springer US
Format: PDF
Kopierschutz: 1 - PDF Watermark

E-Book, Englisch, Band 1586, 429 Seiten, eBook

Reihe: Methods in Molecular Biology

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.

Zielgruppe

Professional/practitioner

Autoren/Hrsg.

Burgess-Brown, Nicola A.

Inhaltsverzeichnis

Recombinant Protein Expression in E. coli: A Historical Perspective.- N- and C-Terminal Truncations to Enhance Protein Solubility and Crystallization: Predicting Protein Domain Boundaries with Bioinformatics Tools.- Harnessing the Profinity eXact TM System for Expression and Purification of Heterologous Proteins in E. coli.- ESPRIT: A Method for Defining Soluble Expression Constructs in Poorly Understood Gene Sequences.- Optimizing Expression and Solubility of Proteins in E. coli Using Modified Media and Induction Parameters.- Optimization of Membrane Protein Production Using Titratable Strains of E. coli.- Optimizing E. coli-Based Membrane Protein Production Using Lemo21(DE3) or pReX and GFP-Fusions.- High Yield of Recombinant Protein in Shaken E. coli Cultures with Enzymatic Glucose Release Medium EnPresso B.- A Generic Protocol for Purifying Disulphide-Bonded Domains and Random Protein Fragments Using FusionProteins with SUMO3 and Cleavage by SenP2 Protease.- A Strategy for Production of Correctly Folded Disulfide-Rich Peptides in the Periplasm of E. coli.- Split GFP Complementation as Reporter of Membrane Protein Expression and Stability in E. coli: A Tool to Engineer Stability in a LAT Transporter.- Acting on Folding Effectors to Improve Recombinant Protein Yields and Functional Quality.- Protein Folding Using a Vortex Fluidic Device.- Removal of Affinity Tags with TEV Protease.- Generation of Recombinant N -Linked Glycoproteins in E. coli.- Production of Protein Kinases in E. coli.- Expression of Prokaryotic Integral Membrane Proteins in E. coli.- Multiprotein Complex Production in E. coli: The SecYEG-SecDFYajC-YidC Holotranslocon.- Membrane Protein Production in E. coli Lysates in Presence of Pre-Assembled Nanodiscs.- Not Limited to E. coli: Versatile Expression Vectors for Mammalian Protein Expression.- A Generic Protocol for Intracellular Expression of Recombinant Proteins in Bacillus subtilis.-  In Vivo Biotinylation of Antigens in E. coli.- Cold-Shock Expression System in E. coli for Protein NMR Studies.- High-Throughput Production of Proteins in E. coli for Structural Studies.- Mass Spectrometric Analysis of Proteins.- How to Determine Interdependencies of Glucose and Lactose Uptake Rates for Heterologous Protein Production with E. coli.- Interfacing Biocompatible Reactions with Engineered Escherichia coli.