Protein Engineering for Therapeutics, Part B

Volume in series

Preface

Stapled Peptides for Intracellular Drug Targets

1. Introduction

2. All-Hydrocarbon Stapled α-Helical Peptides

3. The Design of Stapled Peptides

4. Stapled Peptide Synthesis

5. Olefin Metathesis

6. N-terminal and Internal Modifications

7. Cleavage of Stapled Peptides from the Solid Support

8. Stapled Peptide Purification

9. Biophysical Characterization

10. Cell Permeability

11. In Vitro Target Interaction and Activity Assays

12. In Vivo Efficacy

13. Strategies for Stapled Peptide Optimization

14. Summary

Acknowledgments

Mapping of Vascular ZIP Codes by Phage Display

1. Introduction

2. Methods

3. Concluding Remarks and Perspectives

Acknowledgments

Engineering Cyclic Peptide Toxins

1. Introduction

2. Peptide Design

3. Peptide Synthesis

4. Structural Analysis

5. Stability Assays

6. Special Considerations for Cyclic Peptides

Acknowledgments

Peptide Discovery Using Bacterial Display and Flow Cytometry

1. Introduction

2. Protocols

3. Conclusions

Acknowledgments

Designed Ankyrin Repeat Proteins (DARPins)

1. Introduction

2. Applications of DARPins

3. Protocols for DARPins in Biomedical Applications

Acknowledgment

Target-Binding Proteins Based on the 10th Human Fibronectin Type III Domain (10Fn3)

1. Introduction

2. Library Design

3. Choice of Selection Platform

4. Phage Display, mRNA Display, and Yeast-Surface Display of 10Fn3-Based Libraries

5. Production

6. Conclusion

Acknowledgments

Anticalins

1. Introduction

2. Cloning and Expression of Lipocalins and Anticalins in E. coli

3. Construction of a Genetic Anticalin Library

4. Preparation and Selection of a Phage Display Library for Anticalins

5. Preparation and Selection of a Bacterial Surface Display Library for Anticalins

6. Colony Screening for Anticalins with Specific Target-Binding Activity

7. Screening for Anticalins with Specific Target-Binding Activity Using Microtiter Plate Expression in E. coli

8. Measuring Target Affinity of Anticalins in an ELISA

9. Measuring Target Affinity of Anticalins via Surface Plasmon Resonance

10. Application of Anticalins in Biochemical Research and Drug Development

T Cell Receptor Engineering

1. Introduction

2. Stability and Affinity Engineering of T Cell Receptors by Yeast Surface Display

3. Affinity Engineering and Selection of T Cell Receptors by T Cell Display

4. Expression, Purification, and Applications of Soluble scTv Proteins

5. Recipes for Media and Buffers

Acknowledgments

Engineering Knottins as Novel Binding Agents

1. Introduction

2. Knottins as Scaffolds for Engineering Molecular Recognition

3. Engineering Knottins by Yeast Surface Display

4. Knottin Library Construction

5. Screening Yeast-Displayed Knottin Libraries

6. Knottin production by chemical synthesis or recombinant expression

7. Cell binding assays

8. Summary

Acknowledgments

Practical Theoretic Guidance for the Design of Tumor-Targeting Agents

1. Introduction

2. What Molecular Size Is Best for Tumor Uptake?

3. Will Targeting Increase Nanoparticle Accumulation in a Tumor?

4. How Does Affinity Affect Biodistribution?

5. What Dose Is Necessary in Order to Overcome the “Binding Site Barrier”?

6. Conclusions

Reengineering Biopharmaceuticals for Targeted Delivery Across the Blood–Brain Barrier

1. Introduction

2. Blood–Brain Barrier Receptor-Mediated Transport and Molecular Trojan Horses

3. Reengineering Recombinant Proteins for Targeted Brain Delivery

4. Genetic Engineering of Expression Plasmid DNA Encoding IgG Fusion Proteins

5. Pharmacokinetics and Brain Uptake of IgG Fusion Proteins

6. CNS Pharmacological Effects of IgG Fusion Proteins

7. Immune Response Against IgG Fusion Proteins

8. Summary

Engineering and Identifying Supercharged Proteins for Macromolecule Delivery into Mammalian Cells

1. Introduction

2. Methods

3. Conclusion