Recombinant Protein Protocols: Detection and Isolation / Edition 1

by Rocky S. Tuan

Overview

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 applications 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 Prools 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 prools 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.

Product Details

ISBN-13:	9780896034815
Publisher:	Springer-Verlag New York, LLC
Publication date:	03/05/1997
Series:	Methods in Molecular Biology , #63
Edition description:	1997
Pages:	448
Product dimensions:	6.10(w) x 9.25(h) x 0.05(d)

About the Author

Tuan, Rocky S. (Thomas Jefferson University)

The contributors represent the specialties of gene therapy, microbiology, biochemistry, molecular genetics, and biotechnology. Most are from universities, research institutes, and biomedical research companies in six countries, including the U.S., Sweden, and Germany. Institutions prominently represented include Baxter Healthcare Corp, Auburn Univ, Thomas Jefferson Univ, Oregon State Univ, Univ of Pennsylvania, and Boston Univ.

Table of Contents

Overview of Experimental Strategies on the Detection and Isolation of Recombinant Proteins and Their Applications.- Detection and Isolation of Expressed Proteins.- Reporter Systems.- Detection of Recombinant Protein Based on Reporter Enzyme Activity: Chloramphenicol Acetyltransferase.- Human Placental Alkaline Phosphatase as a Marker for Gene Expression.- Use of Secreted Alkaline Phosphatase as a Reporter of Gene Expression in Mammalian Cells.- Detection of—-Galactosidase and—-Glucuronidase Using Chemiluminescent Reporter Gene Assays.- Chemiluminescent Immunoassay for the Detection of Chloramphenicol Acetyltransferase and Human Growth Hormone Reporter Proteins.- Detection and Selection of Cultured Cells Secreting Recombinant Product by Soft Agar Cloning and Antibody Overlay.- Detection and Isolation of Recombinant Protein Based on Binding Affinity Reporter.- Detection and Isolation of Recombinant Proteins Based on Binding Affinity of Reporter: Protein A.- Expression and Purification of Recombinant Streptavidin-Containing Chimeric Proteins.- Bacterial Expression, Purification, and Potential Use of His-Tagged GAL4 Fusion Proteins.- Detection of Expressed Recombinant Protein Based on Multidrug Resistance: P-Glycoprotein.- Detection and Isolation of Recombinant Proteins from Mammalian Cells by Immunoaffinity Chromatography: p53.- Yeast GAL4 Two-Hybrid System.- Alternative Yeast Two-Hybrid Systems.- Detection of Heterologous Gs-Coupled Receptor Activity in LLC-PK1 Cells Based on Expression of Urokinase-Type Plasminogen Activator.- Detection of Recombinant Gene Expression In Situ.- Hishemical and Fluorochrome-Based Detection of—-Galactosidase.- Combined, Sequential In Situ Hybridization and Immunohishemistry on the Same Tissue Section.- Whole Mount In SituHybridization to Embryos and Embryonic Tissues.- Whole-Mount In Situ Hybridization for Developing Chick Embryos Using Digoxygenin-Labeled RNA Probes.- Methods for Double Detection of Gene Expression.- In Situ PCR.- Expression and Detection of Green Fluorescent Protein (GFP).- Applications of Recombinant Gene Expression.- Transgenic Animals as Bioreactors for Expression of Recombinant Proteins.- Artificial Cells for Bioencapsulation of Cells and Genetically Engineered E. coli.- Hyperexpression of a Synthetic Protein-Based Polymer Gene.- Transplantation of Cells in an immunoisolation Device for Gene Therapy.- Delivery of Recombinant HIV-1-Directed Antisense and Ribozyme Genes.- Inducible System Designed for Future Gene Therapy.- Human Gene Therapy.

Editorial Reviews

3 Stars from Doody

Recombinant Gene Expression Protocols and Recombinant Protein Protocols, edited by Rocky S. Tuan, are two well-written books that contain a wealth of information on construction of vectors for expression of recombinant proteins and for the analysis of the construction vectors in eukaryotic or prokaryotic cells. The purpose of these books is to provide laboratory manuals on the detection and isolation of recombinantly expressed genes. The books are written for any researcher who wants to express or clone a gene -- graduate student through professor. In Recombinant Gene Expression Protocols, expression protocols and detailed background information are presented for many different types of protein expression vectors in E. coli, for expression in yeast with GAL4, PGK or GALA inducible expression vectors, for expression in insect and mammalian cells as well as detailed protocols for plant expression and transgenic organism construction. A truly important facet of the book is that so much information is collected in one book; although the book contains many different expression technologies they are presented in a balanced and thoughtful manner. The companion volume, Recombinant Protein Protocols, is a perfect complement to the expression book and includes detailed protocols on detection of recombinant proteins by several methods (e.g., alkaline phosphatase, CAT, His-tagging) as well as protocols for in situ detection and an excellent section on applications of recombinant gene expression. I would highly recommend this series of books to researchers interested in cloning andexpressing and analyzing their gene of interest using molecular biology techniques.

Philip M. Sass

Experienced researchers describe their techniques for a large number of molecular tags and labels, including enzymes and ligand-binding moieties. They also report on detecting interactive proteins and alterations in cellular activity that are mediated by gene expression, and the "in situ" detection of gene expression. Applications such as gene therapy and transgenic animals are also surveyed. The cookbook style-step-by-step recipes are designed to facilitate reproducible results. A companion volume to "Recombinant Gene Expression Protocols". Plastic comb binding Annotation c. by Book News, Inc., Portland, Or.

Booknews

Reviewer: Philip M. Sass, PhD (University of Illinois at Chicago College of Medicine)
Description: Recombinant Gene Expression Protocols and Recombinant Protein Protocols, edited by Rocky S. Tuan, are two well-written books that contain a wealth of information on construction of vectors for expression of recombinant proteins and for the analysis of the construction vectors in eukaryotic or prokaryotic cells.
Purpose: The purpose of these books is to provide laboratory manuals on the detection and isolation of recombinantly expressed genes.
Audience: The books are written for any researcher who wants to express or clone a gene — graduate student through professor.
Features: In Recombinant Gene Expression Protocols, expression protocols and detailed background information are presented for many different types of protein expression vectors in E. coli, for expression in yeast with GAL4, PGK or GALA inducible expression vectors, for expression in insect and mammalian cells as well as detailed protocols for plant expression and transgenic organism construction. A truly important facet of the book is that so much information is collected in one book; although the book contains many different expression technologies they are presented in a balanced and thoughtful manner. The companion volume, Recombinant Protein Protocols, is a perfect complement to the expression book and includes detailed protocols on detection of recombinant proteins by several methods (e.g., alkaline phosphatase, CAT, His-tagging) as well as protocols for in situ detection and an excellent section on applications of recombinant gene expression.
Assessment: I would highly recommend this series of books to researchers interested in cloning and expressing and analyzing their gene of interest using molecular biology techniques.

Doody's Review Service