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In E. coli Gene Expression Protocol s, Peter E. Vaillancourt, explores the most popular and newly emerging methodologies to exploit the advantages of using E. coli as a host organism for expressing recombinant proteins. The subject matter in this text is displayed in two categories: 1) the use of E. coli vectors and strains for production of pure, functional protein, and 2) the use of E. coli as host for the functional screening of large collections of proteins or peptides. Clear, concise protocols for state-of-the-art techniques relevant to this rapidly emerging field are provided with step-by-step insturctions. This volume will serve as a vehicle to facilitate the widespread use of these improved techniques, and thereby contribute to the rapid advancement of the field. Not only does the text explore in great detail revolutionary findings in protein expression, protein purification, gene discovery, proteomics and protein-protein interactions, it also gives insight to expression vectors, expression strains, phage displays, and two-hybrid systems.These methods and protocols will be of use to researchers over a wide range of disciplines as well as pharmacologists involved in clinical trials, students and practicing physicians. The text will serve as an invaluable reference to academic and industrial biochemists, molecular and cell biologists whose research requires the physical analysis of purified cloned proteins. Scientists interested in new systems for the discovery of interacting proteins and peptides will come to treasure the extensive knowledge contained in this book, and how it will assist them in their research.
Cold-Inducible Promoters for Heterologous Protein Expression.- Dual-Expression Vectors for Efficient Protein Expression in Both E. coli and Mammalian Cells.- A Dual-Expression Vector Allowing Expression in E. coli and P. pastoris, Including New Modifications.- Purification of Recombinant Proteins from E. coli by Engineered Inteins.- Calmodulin as an Affinity Purification Tag.- Calmodulin-Binding Peptide as a Removable Affinity Tag for Protein Purification.- Maltose-Binding Protein as a Solubility Enhancer.- Thioredoxin and Related Proteins as Multifunctional Fusion Tags for Soluble Expression in E. coli.- Discovery of New Fusion Protein Systems Designed to Enhance Solubility in E. coli.- Assessment of Protein Folding/Solubility in Live Cells.- Improving Heterologous Protein Folding via Molecular Chaperone and Foldase Co-Expression.- High-Throughput Purification of PolyHis-Tagged Recombinant Fusion Proteins.- Co-Expression of Proteins in E. coli Using Dual Expression Vectors.- Small-Molecule Affinity-Based Matrices for Rapid Protein Purification.- Use of tRNA-Supplemented Host Strains for Expression of Heterologous Genes in E. coli.- Screening Peptide/Protein Libraries Fused to the ? Repressor DNA-Binding Domain in E. coli Cells.- Studying Protein-Protein Interactions Using a Bacterial Two-Hybrid System.- Using Bio-Panning of FLITRX Peptide Libraries Displayed on E. coli Cell Surface to Study Protein-Protein Interactions.- Use of Inteins for the In Vivo Production of Stable Cyclic Peptide Libraries in E. coli.- Hyperphage.- Combinatorial Biosynthesis of Novel Carotenoids in E. coli.- Using Transcriptional-Based Systems for In Vivo Enzyme Screening.- Identification of Genes Encoding Secreted Proteins Using Mini-OphoA Mutagenesis.
"...many will find it extremely useful to have this book on hand when trying to achieve high levels of pure, soluble recombinant protein; or incorporating an E. coli expression step into the procedures of functional genomics, proteomics and protein engineering." - Microbiology Today