This volume of Methods in Enzymology looks at Protein Engineering for Therapeutics. The chapters provide an invaluable resource for academics, researchers and students alike. With an international board of authors, this volume is split into sections that cover subjects such as Peptides, and Scaffolds
1. 1. Introduction 2. 2. All-Hydrocarbon Stapled α-Helical Peptides 3. 3. The Design of Stapled Peptides 4. 4. Stapled Peptide Synthesis 5. 5. Olefin Metathesis 6. 6. N-terminal and Internal Modifications 7. 7. Cleavage of Stapled Peptides from the Solid Support 8. 8. Stapled Peptide Purification 9. 9. Biophysical Characterization 10. 10. Cell Permeability 11. 11. In Vitro Target Interaction and Activity Assays 12. 12. In Vivo Efficacy 13. 13. Strategies for Stapled Peptide Optimization 14. 14. Summary 15. Acknowledgments 16. Mapping of Vascular ZIP Codes by Phage Display 17. 1. Introduction 18. 2. Methods 19. 3. Concluding Remarks and Perspectives 20. Acknowledgments 21. Engineering Cyclic Peptide Toxins 22. 1. Introduction 23. 2. Peptide Design 24. 3. Peptide Synthesis 25. 4. Structural Analysis 26. 5. Stability Assays 27. 6. Special Considerations for Cyclic Peptides 28. Acknowledgments 29. Peptide Discovery Using Bacterial Display and Flow Cytometry 30. 1. Introduction 31. 2. Protocols 32. 3. Conclusions 33. Acknowledgments 34. Designed Ankyrin Repeat Proteins (DARPins) 35. 1. Introduction 36. 2. Applications of DARPins 37. 3. Protocols for DARPins in Biomedical Applications 38. Acknowledgment 39. Target-Binding Proteins Based on the 10th Human Fibronectin Type III Domain (10Fn3) 40. 1. Introduction 41. 2. Library Design 42. 3. Choice of Selection Platform 43. 4. Phage Display, mRNA Display, and Yeast-Surface Display of 10Fn3-Based Libraries 44. 5. Production 45. 6. Conclusion 46. Acknowledgments 47. Anticalins 48. 1. Introduction 49. 2. Cloning and Expression of Lipocalins and Anticalins in E. coli 50. 3. Construction of a Genetic Anticalin Library 51. 4. Preparation and Selection of a Phage Display Library for Anticalins 52. 5. Preparation and Selection of a Bacterial Surface Display Library for Anticalins 53. 6. Colony Screening for Anticalins with Specific Target-Binding Activity 54. 7. Screening for Anticalins with Specific Target-Binding Activity Using Microtiter Plate Expression in E. coli 55. 8. Measuring Target Affinity of Anticalins in an ELISA 56. 9. Measuring Target Affinity of Anticalins via Surface Plasmon Resonance 57. 10. Application of Anticalins in Biochemical Research and Drug Development 58. T Cell Receptor Engineering 59. 1. Introduction 60. 2. Stability and Affinity Engineering of T Cell Receptors by Yeast Surface Display 61. 3. Affinity Engineering and Selection of T Cell Receptors by T Cell Display 62. 4. Expression, Purification, and Applications of Soluble scTv Proteins 63. 5. Recipes for Media and Buffers 64. Acknowledgments 65. Engineering Knottins as Novel Binding Agents 66. 1. Introduction 67. 2. Knottins as Scaffolds for Engineering Molecular Recognition 68. 3. Engineering Knottins by Yeast Surface Display 69. 4. Knottin Library Construction 70. 5. Screening Yeast-Displayed Knottin Libraries 71. 6. Knottin production by chemical synthesis or recombinant expression 72. 7. Cell binding assays 73. 8. Summary 74. Acknowledgments 75. Practical Theoretic Guidance for the Design of Tumor-Targeting Agents 76. 1. Introduction 77. 2. What Molecular Size Is Best for Tumor Uptake? 78. 3. Will Targeting Increase Nanoparticle Accumulation in a Tumor? 79. 4. How Does Affinity Affect Biodistribution? 80. 5. What Dose Is Necessary in Order to Overcome the “Binding Site Barrier”? 81. 6. Conclusions 82. Reengineering Biopharmaceuticals for Targeted Delivery Across the Blood–Brain Barrier 83. 1. Introduction 84. 2. Blood–Brain Barrier Receptor-Mediated Transport and Molecular Trojan Horses 85. 3. Reengineering Recombinant Proteins for Targeted Brain Delivery 86. 4. Genetic Engineering of Expression Plasmid DNA Encoding IgG Fusion Proteins 87. 5. Pharmacokinetics and Brain Uptake of IgG Fusion Proteins 88. 6. CNS Pharmacological Effects of IgG Fusion Proteins 89. 7. Immune Response Against IgG Fusion Proteins 90. 8. Summary 91. Engineering and Identifying Supercharged Proteins for Macromolecule Delivery into Mammalian Cells 92. 1. Introduction 93. 2. Methods 94. 3. Conclusion
This volume of Methods in Enzymology looks at Protein Engineering for Therapeutics. The chapters provide an invaluable resource for academics, researchers and students alike. With an international board of authors, this volume is split into sections that cover subjects such as Peptides, and Scaffolds
1. 1. Introduction 2. 2. All-Hydrocarbon Stapled α-Helical Peptides 3. 3. The Design of Stapled Peptides 4. 4. Stapled Peptide Synthesis 5. 5. Olefin Metathesis 6. 6. N-terminal and Internal Modifications 7. 7. Cleavage of Stapled Peptides from the Solid Support 8. 8. Stapled Peptide Purification 9. 9. Biophysical Characterization 10. 10. Cell Permeability 11. 11. In Vitro Target Interaction and Activity Assays 12. 12. In Vivo Efficacy 13. 13. Strategies for Stapled Peptide Optimization 14. 14. Summary 15. Acknowledgments 16. Mapping of Vascular ZIP Codes by Phage Display 17. 1. Introduction 18. 2. Methods 19. 3. Concluding Remarks and Perspectives 20. Acknowledgments 21. Engineering Cyclic Peptide Toxins 22. 1. Introduction 23. 2. Peptide Design 24. 3. Peptide Synthesis 25. 4. Structural Analysis 26. 5. Stability Assays 27. 6. Special Considerations for Cyclic Peptides 28. Acknowledgments 29. Peptide Discovery Using Bacterial Display and Flow Cytometry 30. 1. Introduction 31. 2. Protocols 32. 3. Conclusions 33. Acknowledgments 34. Designed Ankyrin Repeat Proteins (DARPins) 35. 1. Introduction 36. 2. Applications of DARPins 37. 3. Protocols for DARPins in Biomedical Applications 38. Acknowledgment 39. Target-Binding Proteins Based on the 10th Human Fibronectin Type III Domain (10Fn3) 40. 1. Introduction 41. 2. Library Design 42. 3. Choice of Selection Platform 43. 4. Phage Display, mRNA Display, and Yeast-Surface Display of 10Fn3-Based Libraries 44. 5. Production 45. 6. Conclusion 46. Acknowledgments 47. Anticalins 48. 1. Introduction 49. 2. Cloning and Expression of Lipocalins and Anticalins in E. coli 50. 3. Construction of a Genetic Anticalin Library 51. 4. Preparation and Selection of a Phage Display Library for Anticalins 52. 5. Preparation and Selection of a Bacterial Surface Display Library for Anticalins 53. 6. Colony Screening for Anticalins with Specific Target-Binding Activity 54. 7. Screening for Anticalins with Specific Target-Binding Activity Using Microtiter Plate Expression in E. coli 55. 8. Measuring Target Affinity of Anticalins in an ELISA 56. 9. Measuring Target Affinity of Anticalins via Surface Plasmon Resonance 57. 10. Application of Anticalins in Biochemical Research and Drug Development 58. T Cell Receptor Engineering 59. 1. Introduction 60. 2. Stability and Affinity Engineering of T Cell Receptors by Yeast Surface Display 61. 3. Affinity Engineering and Selection of T Cell Receptors by T Cell Display 62. 4. Expression, Purification, and Applications of Soluble scTv Proteins 63. 5. Recipes for Media and Buffers 64. Acknowledgments 65. Engineering Knottins as Novel Binding Agents 66. 1. Introduction 67. 2. Knottins as Scaffolds for Engineering Molecular Recognition 68. 3. Engineering Knottins by Yeast Surface Display 69. 4. Knottin Library Construction 70. 5. Screening Yeast-Displayed Knottin Libraries 71. 6. Knottin production by chemical synthesis or recombinant expression 72. 7. Cell binding assays 73. 8. Summary 74. Acknowledgments 75. Practical Theoretic Guidance for the Design of Tumor-Targeting Agents 76. 1. Introduction 77. 2. What Molecular Size Is Best for Tumor Uptake? 78. 3. Will Targeting Increase Nanoparticle Accumulation in a Tumor? 79. 4. How Does Affinity Affect Biodistribution? 80. 5. What Dose Is Necessary in Order to Overcome the “Binding Site Barrier”? 81. 6. Conclusions 82. Reengineering Biopharmaceuticals for Targeted Delivery Across the Blood–Brain Barrier 83. 1. Introduction 84. 2. Blood–Brain Barrier Receptor-Mediated Transport and Molecular Trojan Horses 85. 3. Reengineering Recombinant Proteins for Targeted Brain Delivery 86. 4. Genetic Engineering of Expression Plasmid DNA Encoding IgG Fusion Proteins 87. 5. Pharmacokinetics and Brain Uptake of IgG Fusion Proteins 88. 6. CNS Pharmacological Effects of IgG Fusion Proteins 89. 7. Immune Response Against IgG Fusion Proteins 90. 8. Summary 91. Engineering and Identifying Supercharged Proteins for Macromolecule Delivery into Mammalian Cells 92. 1. Introduction 93. 2. Methods 94. 3. Conclusion
