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Protein Engineering - 1st Edition - ISBN: 9780123724854, 9780323150309

Protein Engineering

1st Edition

Applications In Science, Medicine, and Industry

Editor: Raghupathy Sarma
eBook ISBN: 9780323150309
Imprint: Academic Press
Published Date: 28th October 1986
Page Count: 440
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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


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




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© Academic Press 1986
28th October 1986
Academic Press
eBook ISBN:

About the Editor

Raghupathy Sarma

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