Recombinant Gene Expression

Since newly created beings are often perceived as either wholly good or bad, the genetic alteration of living cells impacts directly on a symbolic meaning deeply imbedded in every culture. During the earlier years of gene expression research, te- nological applications were confined mainly to academic and industrial laboratories, and were perceived as highly beneficial since molecules that were previously unable to be separated or synthesized became accessible as therapeutic agents. Such were the success stories of hormones, antibodies, and vaccines produced in the bacterium Escherichia coli. Originally this bacterium gained fame among humans for being an unwanted host in the intestine, or worse yet, for being occasionally dangerous and pathogenic. H- ever, it was easily identified in contaminated waters during the 19th century, thus becoming a clear indicator of water pollution by human feces. Tamed, cultivated, and easily maintained in laboratories, its fast growth rate and metabolic capacity to adjust to changing environments fascinated the minds of scientists who studied and modeled such complex phenomena as growth, evolution, genetic exchange, infection, survival, adaptation, and further ongene expression. Although at the lower end of the complexity scale, this microbe became a very successful model system and a key player in the fantastic revolution kindled by the birth of recombinant DNA technology.

Includes supplementary material: sn.pub/extras

Klappentext
In this updated and expanded second edition of an established classic, the editors have added critical reviews to a fresh collection of cutting-edge protocols for gene expression in bacteria, fungi, plants, plant cells, animals, and animal cells. The review articles survey new directions in recombinant gene expression research, technique, and application, and point the way to using recombinant gene expression for metabolic engineering and the production of nonprotein molecules. The readily reproducible protocols offer new host-vector systems, such as Antarctic bacteria and moderately halophytic bacteria, vectors for chromosomal editing and metabolic engineering, and information to aid in problem solving, strategy planning, and the promotion of protein folding. There are also novel heterologous gene expression schemes for cell-free systems, plant chloroplasts, microplates, hairy roots, and animal milk, and new developments in consolidated heterologous systems. The protocols follow the successful Methods in Molecular Biology™ series format, each one offering step-by-step laboratory instructions, an introduction outlining the principle behind the technique, lists of equipment and reagents, and tips on troubleshooting and avoiding known pitfalls.
State-of-the-art and forward looking, Recombinant Gene Expression: Reviews and Protocols, Second Edition offers investigators seeking an overview of this critically important field not only the understanding, but also the tools needed to begin producing nonprotein products and altering the central metabolic pathways of cells to enhance heterologous gene expression.

Inhalt
General Issues About Recombinant Gene Expression.- Host Cell Compatibility in Protein Expression.- Production of Recombinant Proteins.- Folding-Promoting Agents in Recombinant Protein Production.- Prokaryotes.- Back to Basics.- ?-Complementation-Enabled T7 Expression Vectors and Their Use for the Expression of Recombinant Polypeptides for Protein Transduction Experiments.- Expression of Recombinant Alkaline Phosphatase Conjugates in Escherichia coli.- Overexpression of Chromosomal Genes in Escherichia coli.- Chromosomal Expression of Foreign and Native Genes From Regulatable Promoters in Escherichia coli.- Plasmid Vectors for Marker-Free Chromosomal Insertion of Genetic Material in Escherichia coli.- Copy-Control pBAC/oriV Vectors for Genomic Cloning.- Copy-Control Tightly Regulated Expression Vectors Based on pBAC/oriV.- Cell-Free Protein Synthesis With Prokaryotic Combined Transcription-Translation.- Genetic Tools for the Manipulation of Moderately Halophilic Bacteria of the Family Halomonadaceae.- Gene Transfer and Expression of Recombinant Proteins in Moderately Halophilic Bacteria.- Recombinant Protein Production in Antarctic Gram-Negative Bacteria.- Fungi.- Recombinant Protein Production in Yeasts.- Controlled Expression of Homologous Genes by Genomic Promoter Replacement in the Yeast Saccharomyces cerevisiae.- High-Throughput Expression in Microplate Format in Saccharomyces cerevisiae.- High-Throughput Expression in Microplate Format in Pichia pastoris.- Multiple Gene Expression by Chromosomal Integration and CRE-loxP-Mediated Marker Recycling in Saccharomyces cerevisiae.- Three Decades of Fungal Transformation.- Three Decades of Fungal Transformation.- Plants and Plant Cells.- Gene Transfer and Expression in Plants.- Production of Recombinant Proteins by HairyRoots Cultured in Plastic Sleeve Bioreactors.- Engineering the Chloroplast Genome for Hyperexpression of Human Therapeutic Proteins and Vaccine Antigens.- New Selection Marker for Plant Transformation.- Enhancer Detection and Gene Trapping as Tools for Functional Genomics in Plants.- Animals and Animal Cells.- Gene Transfer and Expression in Mammalian Cell Lines and Transgenic Animals.- Sustained Heterologous Transgene Expression in Mammalian and Avian Cell Lines.- Inducible Gene Expression in Mammalian Cells and Mice.- Flp-Mediated Integration of Expression Cassettes into FRT-Tagged Chromosomal Loci in Mammalian Cells.- Generation of High-Recombinant-Protein-Producing Chinese Hamster Ovary (CHO) Cells.- Preparation of Recombinant Proteins in Milk.