Protein Engineering

Protein Engineering

Applications In Science, Medicine, and Industry

1st Edition - October 28, 1986

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  • Editor: Raghupathy Sarma
  • eBook ISBN: 9780323150309

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Protein Engineering: Applications in Science, Medicine, and Industry deals with the scientific, medical, and industrial applications of protein engineering. Topics range from protein structure and design to mutant analysis and complex systems. Applications such as production of novel antibiotics, genetic transformation of plants, and genetic engineering of bioinsecticides are described. This book is comprised of 25 chapters and begins with an overview of trends and developments in protein chemistry and their relevance to protein engineering, followed by a discussion on protein sequence data banks. Subsequent chapters explore the design and construction of biologically active peptides, including hormones; structural and functional analysis of thermophile proteins; the conformation of diphtheria toxin; and applications of surface-simulation synthesis in protein molecular recognition. The use of oligonucleotide-directed site-specific mutagenesis in functional analysis of the signal peptide for protein secretion is also considered. The results of studies on the mechanism of membrane fusion are presented. This monograph will serve as a useful guide for those who are already working on protein engineering and those who are about to start research in this field.

Table of Contents

  • Preface

    I Structure and Design

    1 Classical Protein Chemistry in a World of Slicing and Splicing



    2 Protein Sequence Data Banks: The Continuing Search for Related Structures

    I. Introduction

    II. Computer Searching Methods

    III. Nucleotide Binding Sequences

    IV. Concluding Remarks


    3 The Analysis of Homologous Tertiary Structures and the Design of Novel Proteins

    I. Introduction

    II. Engineering Amino Acid Replacements, Insertions, and Deletions

    III. Modeling Homologous Proteins

    IV. Conclusions for Protein Engineering


    4 Structural Implications for Macromolecular Recognition and Redesign

    I. Introduction

    II. Dissection of a Protein Structure

    III. Intermolecular Interactions

    IV. Engineering Principles


    5 The Design and Construction of Biologically Active Peptides, Including Hormones

    I. Introduction

    II. Development of Principles for the Design of Models for Surface-Active Peptides and Proteins

    III. Construction of Amphiphilic Helical Models Apolipoproteins, Peptide Toxins, and Hormones

    IV. The Design of Amphiphilic ß Strands


    6 Structural and Functional Analysis of Thermophile Proteins

    I. Tactics of Thermophiles

    II. Catalytic Properties of Thermophilic Enzymes

    III. Physicochemical Studies

    IV. tRNA as a Model

    V. Molecular Cloning


    7 The Conformation of Diphtheria Toxin: A Protein That Penetrates Membranes at Low pH

    I. Diphtheria Toxin Structure and Function

    II. The Hydrophilic-to-Hydrophobic Switch: Transition pH

    III. The Hydrophilic-to-Hydrophobic Switch: Conformational Changes

    IV. Mechanism of the Conformational Changes

    V. Implications for the Conformation of Other Proteins and Design of Modified Toxins


    8 Design and Total Chemical Synthesis of a Gene for Bovine Rhodopsin

    I. Introduction

    II. Design of the Gene

    III. Synthesis and Assembly of the Gene

    IV. In Vitro Expression of the Synthetic Rhodopsin Gene

    V. Summary


    9 Surface-Simulation Synthesis and Its Applications in Protein Molecular Recognition

    I. Introduction

    II. Development of Surface-Simulation Synthesis

    III. Applications of Surface-Simulation Synthesis

    IV. Conclusions


    II Mutant Analysis

    10 Functional Analysis of the Signal Peptide for Protein Secretion with Use of Oligonucleotide-Directed Site-Specific Mutagenesis

    I. Introduction

    II. The Signal Peptide

    III. Site-Specific Mutagenesis

    IV. Mutant Analysis

    V. Conclusion


    11 Physical Properties of Genetically Defined Synthetic Signal Sequences Suggest Initial Steps in Protein Export

    I. Introduction

    II. Are Conformational Properties of Signal Sequences Correlated with Function?

    III. Are Membrane Interactions of Signal Sequences Correlated with Function?

    IV. Can the Conformational Properties of Signal Sequences Be Related to Their Interactions with Monolayers?

    V. Proposed Model for the Initial Interaction of Signal Sequences with the Membrane


    12 Studies on the Mechanism of Membrane Fusion

    I. Introduction

    II. Structure and Function of Hemagglutinin

    III. Assays for the Low-pH-Induced Conformational Change and the Fusion Activity of the Hemagglutinin Molecule

    IV. Analysis of the Hemagglutinin from a Variant Influenza Virus That Induces Fusion at Elevated pH

    V. Site-Directed Mutagenesis of the Fusion Peptide of Hemagglutinin

    VI. Conclusions


    Expression and Site-Specific Mutagenesis of an Integral Membrane Protein, Bacterio-Opsin

    I. Introduction

    II. Cloning and Expression of Bacterio-Opsin Gene

    III. Purification and Reconstitution of E. coli-Produced Bacterio-Opsin

    IV. Mutagenesis of Bacterio-Opsin Gene

    V. Phenotypes of Bacteriorhodopsin Mutants

    VI. Concluding Remarks


    14 Stability Mutants of Staphylococcal Nuclease: A Correlation between Nuclease Activity in an Agar Gel Assay and Stability to Guanidine Hydrochloride Denaturation

    I. Introduction

    II. Materials and Methods

    III. Results

    IV. Discussion and Conclusions


    15 Mutagenesis of the Arc Repressor Using Synthetic Primers with Random Nucleotide Substitutions

    I. Introduction

    II. Materials and Methods

    III. Results

    IV. Discussion


    16 Investigation of the Structural Roles of Disulfides by Protein Engineering: A Study with T4 Lysozyme

    I. Introduction

    II. Properties of Disulfides in Globular Proteins

    III. T4 Lysozyme

    IV. Expression of T4 Lysozyme Gene in E. coli

    V. Criteria and Choice of a Cross-Linking Site

    VI. Introduction of the 3-97 Disulfide into T4 Lysozyme

    VII. Properties of T4 Lysozyme(BC)

    VIII. Stability toward Irreversible Thermal Inactivation

    IX. How Does the 3-97 Disulfide Stabilize T4 Lysozyme?

    X. Uses of Engineered Disulfides


    17 Genetic Identification of Amino Acid Sequences Influencing Protein Folding

    I. The Coding Aspect of the Folding Problem

    II. Genetic Analysis of Protein Folding

    III. Discussion


    III Complex Systems

    18 Structural Basis for Acetylcholine Receptor Function

    I. Introduction

    II. Primary Structure

    III. Expression of Cloned cDNAs

    IV. Subunit Requirement

    V. Deletion Mapping of Functional Regions

    VI. Mutations in the Acetylcholine Binding Region

    VII. Concluding Remarks


    19 Protein Engineering of Antibody Molecules

    I. Introduction

    II. Stable Transfection of Myeloma Cells

    III. Chimeric Antibodies and Immunoglobulin Exon Shuffling

    IV. Substituting Novel Activities for the Antibody Fc Portion

    V. Summary and Prospect


    20 Proteolytic Processing of the Polio Virus Polyprotein by Two Virus-Encoded Proteinases

    I. Introduction

    II. The Cleavage Sites of the Poliovirus Polyprotein

    III. The Kinetics of Individual Cleavages

    IV. Proteinases Involved in Proteolytic Processing

    V. Is Folding the Major Determinant of Processing?

    VI. Conclusion


    IV Applications

    21 Enzymatic Reactions in Organic Media

    I. Introduction

    II. Lipases in Organic Solvents

    III. Oxidoreductases in Organic Solvents

    IV. Biotechnological Applications


    22 Antibody Targeting of Toxin Polypeptides

    I. Introduction

    II. Monoclonal Antibodies for Breast Cancer Immunotoxins

    III. Toxin and Linker for Breast Cancer Immunotoxins

    IV. Discussion


    23 Production of Novel Antibiotics by Gene Cloning and Protein Engineering

    I. Introduction

    II. Macrolide Antibiotics

    III. Glycopeptide Antibiotics

    IV. β-Lactam Antibiotics

    V. Prospects for the Future


    24 Genetic Transformation of Plants

    I. Introduction

    II. Vector Construction

    III. Disarmed Vector

    IV. Plant Transformation

    V. Gene Expression

    VI. Applications and Needs


    25 Genetic Engineering of Bioinsecticides

    I. Introduction

    II. Bacillus thuringiensis kurstaki

    III. Cloning for a Better Bacillus thuringiensis Product

    IV. Biotoxicity Assays as a Barrier

    V. The Crystal-Toxin Genes of Bacillus thuringiensis kurstaki

    VI. Conclusion



Product details

  • No. of pages: 440
  • Language: English
  • Copyright: © Academic Press 1986
  • Published: October 28, 1986
  • Imprint: Academic Press
  • eBook ISBN: 9780323150309

About the Editor

Raghupathy Sarma

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