Protein Engineering

Preface

I Structure and Design


1 Classical Protein Chemistry in a World of Slicing and Splicing

Text

References


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

I. Introduction

II. Computer Searching Methods

III. Nucleotide Binding Sequences

IV. Concluding Remarks

References


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

References


4 Structural Implications for Macromolecular Recognition and Redesign

I. Introduction

II. Dissection of a Protein Structure

III. Intermolecular Interactions

IV. Engineering Principles

References


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

References


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

References


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

References


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

References


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

References

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

References


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

References


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

References

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

References


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

References


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

I. Introduction

II. Materials and Methods

III. Results

IV. Discussion

References


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

References


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

References

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

References


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

References


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

References

IV Applications


21 Enzymatic Reactions in Organic Media

I. Introduction

II. Lipases in Organic Solvents

III. Oxidoreductases in Organic Solvents

IV. Biotechnological Applications

References


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

References


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

References


24 Genetic Transformation of Plants

I. Introduction

II. Vector Construction

III. Disarmed Vector

IV. Plant Transformation

V. Gene Expression

VI. Applications and Needs

References


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

References

Index