Recombinant Protein Protocols

1. Front Matter

   Pages i-xx

   PDF

2. Introduction

   1. Overview of Experimental Strategies on the Detection and Isolation of Recombinant Proteins and Their Applications

      • Rocky S. Tuan

      Pages 3-7

3. Detection and Isolation of Expressed Proteins

   1. Reporter Systems

      • Debyra Groskreutz, Elaine T. Schenborn

      Pages 11-30

   2. Detection of Recombinant Protein Based on Reporter Enzyme Activity: Chloramphenicol Acetyltransferase

      • Peiyu Lee, Dennis E. Hruby

      Pages 31-40

   3. Human Placental Alkaline Phosphatase as a Marker for Gene Expression

      • Paul Bates, Michael H. Malim

      Pages 41-48

   4. Use of Secreted Alkaline Phosphatase as a Reporter of Gene Expression in Mammalian Cells

      • Steven R. Kain

      Pages 49-60

   5. Detection of β-Galactosidase and β-Glucuronidase Using Chemiluminescent Reporter Gene Assays

      • Corinne E. M. Olesen, John J. Fortin, John C. Voyta, Irena Bronstein

      Pages 61-70

   6. Chemiluminescent Immunoassay for the Detection of Chloramphenicol Acetyltransferase and Human Growth Hormone Reporter Proteins

      • Corinne E. M. Olesen, John C. Voyta, Irena Bronstein

      Pages 71-76

   7. Detection and Selection of Cultured Cells Secreting Recombinant Product by Soft Agar Cloning and Antibody Overlay

      • Marylou G. Gibson, Karmen Hodges, Lauretta Lowther

      Pages 77-83

   8. Detection and Isolation of Recombinant Protein Based on Binding Affinity Reporter

      • Paul Riggs

      Pages 85-101

   9. Detection and Isolation of Recombinant Proteins Based on Binding Affinity of Reporter: Protein A

      • Stefan Ståhl, Per-Åke Nygren, Mathias Uhlén

      Pages 103-118

   10. Expression and Purification of Recombinant Streptavidin-Containing Chimeric Proteins

       • Takeshi Sano, Cassandra L. Smith, Charles R. Cantor

       Pages 119-128

   11. Bacterial Expression, Purification, and Potential Use of His-Tagged GAL4 Fusion Proteins

       • M. Lienhard Schmitz, Patrick A. Baeuerle

       Pages 129-137

   12. Detection of Expressed Recombinant Protein Based on Multidrug Resistance: P-Glycoprotein

       • Ursula A. Germann

       Pages 139-159

   13. Detection and Isolation of Recombinant Proteins from Mammalian Cells by Immunoaffinity Chromatography: p53

       • Jamil Momand, Bahman Sepehrnia

       Pages 161-172

   14. Yeast GAL4 Two-Hybrid System

       • Li Zhu

       Pages 173-196

   15. Alternative Yeast Two-Hybrid Systems

       • Erica A. Golemis, Vladimir Khazak

       Pages 197-218

   16. Detection of Heterologous Gs-Coupled Receptor Activity in LLC-PK1 Cells Based on Expression of Urokinase-Type Plasminogen Activator

       • Luigi Catanzariti, Brian A. Hemmings

       Pages 219-225

4. Detection of Recombinant Gene Expression In Situ

   1. Histochemical and Fluorochrome-Based Detection of β-Galactosidase

      • Ruth Sullivan, Cecilia W. Lo

      Pages 229-246

   2. Combined, Sequential In Situ Hybridization and Immunohistochemistry on the Same Tissue Section

      • Kenneth J. Shepley, Rocky S. Tuan

      Pages 247-256

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.

• Thomas Jefferson University, Philadelphia

  Rocky S. Tuan

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