Engineering PH20 Hyaluronidase via Rational Design for Enhanced Catalytic Activity and Thermostability

목차 (Table of Contents)

• 1. Introduction 1
• 1.1 Hyaluronic acid(HA) 1
• 1.2 Human Hyaluronidase PH20 (HuPH20) 2
• 1.3 Therapeutic Applications and Engineering Challenges of Recombinant Human PH20 3
• 2. Materials and methods 4
• 2.1 Expression plasmids Construction 4
• 2.2 Expression and purification of rHuPH20 and variants in mammalian cell 4
• 2.3 Modeling of AlphaFold2 5
• 2.4 Molecular Docking 5
• 2.5 Sequence Conservation and Mutational Tolerance Analysis 5
• 2.6 Computational Prediction of Thermostabilizing Mutations 6
• 2.7 B-factor Analysis 7
• 2.8 Turbidimetric assay 7
• 2.9 Morgan-elson assay 8
• 2.10 Operational Thermostability (T₅₀) Assay 8
• 2.11 Thermal Stability Assay 8
• 2.12 Structure-Based Interaction and Visualization Analysis 9
• 3. Results 10
• 3.1 Structure-Guided Identification and Refinement of Mutation-Permissive Hotspots 10
• 3.1.1. Conservation of the Catalytic Architecture Across Mammalian PH20 Homologs 10
• 3.1.2. Structural Definition of Second-Shell Mutation-Permissive Residues 11
• 3.2 Consensus- and Substrate-Informed Substitution Design at Refined Hotspot Sites 14
• 3.2.1 Conservation-Guided Refinement of Mutation-Permissive Residues 14
• 3.2.2 Structural Definition of Second-Shell Mutation-Permissive Residues 16
• 3.3 Rational Design and Functional Validation of Single-Point Mutants 17
• 3.4 Structural Dissection of Activity-Enhancing Mutants Using Docking-Based Enzyme–Substrate Complexes 19
• 3.5 Development of rHuPH20 Combination Mutants Derived from Single-Point Mutations 22
• 3.6 Computational Design Strategies for Improving Thermal Stability of rHuPH 25
• 3.7 Validation of Enhanced Thermostability in rHuPH20 Single-Point Mutants 26
• 3.8 Design of Dual-Optimized rHuPH20 Variants with Enhanced Activity and Thermal Stability 31
• 3.9 Further Enhancement of Catalytic Activity and Thermal Stability of rHuPH20 via Advanced Combinatorial Design 34
• 4. Discussion 38
• CONCLUSION 40
• REFERENCES 41
• ABSTRACT IN KOREAN 48