Riassunto
A major success story of modem molecular biology is the development of technologies to clone and express specific genes. Current applications of recombinant gene products cover a wide spectrum, including gene therapy, production of bioactive pharmaceuticals, synthesis of novel biopolymers, agriculture and animal husbandry, and so on. Inherent in bringing these appli cations to fruition is the need to design "expression constructs" that will per mit the ready and specific detection and isolation of the defined recombinant gene products. Recombinant Protein Protocols grows out of the need for a laboratory manual on the detection and isolation of recombinantly expressed genes that covers both the background information and the practical laboratory recipes for these analyses. In this book, detailed and contemporary protocols are col lected to provide the reader with a wide-ranging number of methodologies to enhance the detection and isolation of their gene product(s) of interest. A large number of molecular tags and labels and their usage are described, including enzymes, ligand-binding moieties, immunodetectable molecules, as well as methods to detect interactive proteins, and gene expression-mediated alter ations in cellular activity. Chapters on in situ detection of gene expression deal with technologies that are currently being applied to the study of gene function and activity. Highlights of applications for recombinant gene expres sion technologies are provided to give readers exciting perspectives on the future of such technologies.
Contenuti
Part I. Introduction. Overview of Experimental Strategies for the Detection and Isolation of Recombinant Proteins and Their Applications, Rocky S. Tuan. Part II. Detection and Isolation of Expressed Proteins. Reporter Systems, Debyra J. Groskreutz and Elaine T. Schenborn. Detection of Recombinant Protein Based on Reporter Enzyme Activity: Chloramphenicol Acetyltransferase, Peiyu Lee and Dennis E. Hruby. Human Placental Alkaline Phosphatase as a Marker for Gene Expression, Paul Bates and Michael H. Malim. Use of Secreted Alkaline Phosphatase as a Reporter of Gene Expression in Mammalian Cells, Steven R. Kain. Detection of b-Galactosidase and b-Glucuronidase Using Chemiluminescent Reporter Gene Assays, Corinne E. M. Olesen, John J. Fortin, John C. Voyta, and Irena Bronstein. Chemiluminescent Immunoassay for the Detection of Chloramphenicol Acetyltransferase and Human Growth Hormone Reporter Genes, Corinne E. M. Olesen, John C. Voyta, and Irena Bronstein. Detection and Selection of Cultured Cells Secreting Recombinant Product by Soft Agar Cloning and Antibody Overlay, Marylou G. Gibson, Karmen Hodges, and Lauretta Lowther. Detection and Isolation of Recombinant Protein Based on Binding Affinity Reporter: Maltose Binding Protein, Paul Riggs. Detection and Isolation of Recombinant Proteins Based on Binding Affinity of Reporter: Protein A, Stefan Ståhl, Per-Åke Nygren, and Mathias Uhlén. Expression and Purification of Recombinant Streptavidin-Containing Chimeric Proteins, Takeshi Sano, Cassandra L. Smith, and Charles R. Cantor. Bacterial Expression, Purification, and Potential Use of His-Tagged GAL4 Fusion Proteins, M. Lienhard Schmitz and Patrick A. Baeuerle. Detection of Expressed Recombinant Protein Based on Multidrug Resistance: P-Glycoprotein, Ursula A. Germann. Detection and Isolation of Recombinant Proteins from Mammalian Cells by Immunoaffinity Chromatography: p53, Jamil Momand and Bahman Sepehrnia. Yeast GAL4 Two-Hybrid System: A Genetic System to IdentifyProteins That Interact with a Target Protein, Li Zhu. Alternative Yeast Two-Hybrid Systems: The Interaction Trap and Interaction Mating, Erica A. Golemis and Vladimir Khazak. Detection of Heterologous Gs-Coupled Receptor Activity in LLC-PK1 Cells Based on Expression of Urokinase-Type Plasminogen Activator, Luigi Catanzariti and Brian A. Hemmings. Part III. Detection of Recombinant Gene Expression In Situ. Histochemical and Detection of b-Galactosidase, Ruth Sullivan and Cecilia W. Lo. Combined, Sequential In Situ Hybridization and Immunohistochemistry on the Same Tissue Section, Kenneth J. Shepley and Rocky S. Tuan. Whole Mount In Situ Hybridization to Embryos and Embryonic Tissues, Ronald A. Conlon. Whole-Mount In Situ Hybridization for Developing Chick Embryos Using Digoxygenin-Labeled RNA Probes, Christopher W. Hsu and Rocky S. Tuan. Methods for Double Detection of Gene Expression: Combined In Situ Hybridization and Immunocytochemistry or Histochemistry, Ronald A. Conlon. In Situ PCR: Current Protocol, Omar Bagasra, Muhammad Mukhtar, Farida Shaheen, and Roger J. Pomerantz. Expression and Detection of Green Fluorescent Protein (GFP), Steven R. Kain and Paul Kitts. Part IV. Applications of Recombinant Gene Expression. Transgenic Animals as Bioreactors for Expression of Recombinant Proteins, Jaspal S. Khillan. Artificial Cells for Bioencapsulation of Cells and Genetically Engineered E. coli: For Cell Therapy, Gene Therapy, and Removal of Urea and Ammonia, Thomas M. S. Chang and Satya Prakash. Hyperexpression of a Synthetic Protein-Based Polymer Gene, Henry Daniell, Chittibabu Guda, David T. McPherson, Xiaorong Zhang, Jie Xu, and Dan W. Urry. Transplantation of Cells in an Immunoisolation Device for Gene Therapy, Victoria E. Carr-Brendel, Robin L. Geller, Tracy J. Thomas, Daniel R. Boggs, Susan K. Young, JoAnne Crudele, Laura A. Martinson, David A. Maryanov, Robert C. Johnson, and James H. Brauker. Delivery of Recombinant HIV-1-Directed Antisense and Ribozyme Genes
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