Therapeutic Proteins : Methods and Protocols 2720

This volume covers the latest key aspects of therapeutic protein applications. Chapters in this book cover topics such as the discovery, production, and conjugation of protein-proteins with discussions on the direction of future development and advancements; ways to use these engineering proteins for therapeutic and vaccine applications; and the use of modified protein nanocarriers. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and practical, Therapeutic Proteins: Methods and Protocols is a valuable resource for any researcher who are interested in learning more about the field of therapeutic proteins. Preface Contents Contributors Part I: Discovery, Production, and Conjugation of Therapeutic Proteins Chapter 1: Optimizing Cell-Free Protein Synthesis for Antimicrobial Protein Production 1 Introduction 2 Materials 2.1 2x YTPG Medium 2.2 S30 Buffer 2.3 CFPS Reagents 2.4 Strains and Plasmids 2.5 Inhibition Zone Agar Plates 3 Methods 3.1 Harvesting Bacterial Cultures 3.2 Extract Preparation Via Sonication 3.3 Cell-Free Protein Synthesis Reaction 3.4 sfGFP Quantification 3.5 Purification of E3 Immunity Protein 3.6 Coexpression of E3 Colicin and E3 Immunity Protein 3.7 Autoradiography 3.8 Inhibition Zone Tests 4 Notes References Chapter 2: Design and Construction of Antibody Fusion Proteins Incorporating Variable New Antigen Receptor (VNAR) Domains 1 Introduction 2 Materials 2.1 Construct Design 2.2 DNA Prep 2.3 Transient Transfection 2.4 Protein Purification 3 Methods 3.1 Construct Design 3.2 DNA Prep 3.3 Transient Transfection 3.3.1 Small-Scale Transfection 3.3.2 Protein Miniprep 3.3.3 Large-Scale Transfection 3.4 Protein Purification 4 Notes References Chapter 3: Selective and Site-Specific Incorporation of Nonstandard Amino Acids Within Proteins for Therapeutic Applications 1 Introduction 2 Materials 3 Methods 3.1 Identification of an AARS and Media Conditions for Optimal, Selective Protein Titer Using nsAA of Interest 3.2 Posttranslational Proofreading for Improving Purity of N-Terminally Incorporated nsAAs or Identification of Alternative A... 3.3 Site Selection for nsAA Incorporation Within Protein of Interest 3.4 Assessment of Efficiency of nsAA Incorporation During Therapeutic Protein Overexpression 3.5 Purification and Characterization of Therapeutic Protein with nsAA 4 Notes References Chapter 4: An Approach for Antigen-Agnostic Identification of Virus-Like Particle-Displayed Epitopes that Engage Specific Anti... 1 Introduction 2 Materials 2.1 Round 1 Affinity Selection 2.2 Reverse Transcription 2.3 Polymerase Chain Reaction 2.4 PCR Purification 2.5 Digest of PCR Product and Vector 2.6 Gel Extraction or PCR Cleanup 2.7 Ligation 2.8 Ethanol Precipitation 2.9 Transformation and Plasmid Preparation 2.10 VLP Production 2.11 VLP Isolation and Purification 2.12 Rounds 2-5 Affinity Selections 3 Methods 3.1 Round 1 Selection 3.2 Reverse Transcription 3.3 Polymerase Chain Reaction 3.4 PCR Cleanup 3.5 Digest of PCR Product and Vector 3.6 Gel Extraction or PCR Cleanup 3.7 Ligation 3.8 Ethanol Precipitation 3.9 Transformation and Plasmid Preparation 3.10 VLP Production 3.11 VLP Isolation and Purification 3.12 Rounds 2-5 Selection 3.13 Deep Sequencing 4 Notes 5 Discussion and Results References Chapter 5: Split-Protein Therapeutic Platforms: Identifying Binder Pairs 1 Introduction 2 Materials 2.1 Computational Platforms 2.2 Reagents for Empirical Testing 3 Methods 3.1 In Silico Predictions 3.1.1 Predict Structures of Binders and/or Target Antigen 3.1.2 Predict Binding Epitope and Interactions 3.2 Empirical Testing 3.2.1 Cell-Based Screening 3.2.2 Recombinant Protein-Based Screening 4 Notes References Chapter 6: Metal-Mediated Ligand Affinity Chemistry (MLAC) 1 Introduction 2 Materials 2.1 Chemical Synthesis of MLAC Reagent 2.2 Sample Preparation for Mass Spectrometry 2.3 Mass Spectrometry Analysis 3 Methods (Fig. 1) 3.1 Choose a Protein of Interest 3.2 Find a Suitable Affinity Ligand 3.3 Conjugate the Affinity Ligand to the Gold(III) Warhead 3.4 Optimize Conditions for Protein Modification Using Short Model Peptides (Recommended) 3.5 Incubate Protein of Interest with MLAC Reagent (KRASi-Au-1) 3.6 Perform ESI-LC-MS/MS to Find the Specific Modification Site 3.6.1 Sample Preparation for Mass Spectrometry 3.6.2 Mass Spectrometry Analysis 4 Notes References Part II: Engineering Proteins for Therapeutic and Vaccine Applications Chapter 7: Recombinant Elastin-Based Bioelastomers for Biomedical Applications 1 Introduction 2 Materials 2.1 Expression 2.2 Purification 2.3 DOPA Conversion 3 Methods 3.1 Protein Expression 3.2 Purification 3.2.1 Cell Lysis 3.2.2 Salting Out 3.2.3 Inverse Transition Cycles 3.2.4 Protein Processing 3.3 LCST Determination 3.4 ELP Conversion of Tyrosine Residues to DOPA 4 Notes References Chapter 8: Engineering Hepatitis B Virus (HBV) Protein Particles for Therapeutic Delivery 1 Introduction 1.1 Engineering HBV-SpyCatcher VLPs 1.2 Modular Conjugation 1.3 Multi-Functionalization for Cancer Cell Targeting and Therapy 2 Materials 2.1 Production of HBV-SpyCatcher VLPs and SpyTag Fusion Proteins 2.1.1 Culture Medium 2.1.2 Protein Expression and Cell Lysis 2.2 SpyTag/SpyCatcher Bioconjugation 2.2.1 SpyTag/SpyCatcher Reaction 2.2.2 Inverse Transition Cycling (ITC) 2.2.3 SDS-PAGE 2.2.4 Equipment 2.3 Cell Culture 2.4 VLP Delivery 2.5 Flow Cytometry 2.5.1 Equipment 2.6 MTT Assay 3 Methods 3.1 Production of HBV-SpyCatcher VLPs and SpyTag Fusion Proteins 3.1.1 Protein Expression and Cell Lysis 3.1.2 Purification of HBV-SpyCatcher VLPs 3.1.3 Purification of SpyTag Fusion Proteins 3.2 SpyTag/SpyCatcher Conjugation 3.2.1 ELP Purification 3.2.2 SDS-PAGE 3.3 Mammalian Cell Culture 3.4 VLP Delivery and Flow Cytometry 3.5 MTT Viability Assay References Chapter 9: Design and Purification of Tag/Catcher AP205-Based Capsid Virus-Like Particle Vaccines 1 Introduction 2 Materials 2.1 Design of Vaccine Antigen 2.2 Expression of AP205 cVLPs in E.coli 2.3 Purification of AP205 cVLPs 2.3.1 Lysis of Bacterial Cells 2.3.2 Purification by Ultracentrifugation 2.4 Small-Scale Test Coupling of Antigen to cVLPs 2.5 Vaccine Production 2.5.1 Removal of Endotoxin 2.5.2 Final Vaccine Preparation 3 Methods 3.1 Design of Vaccine Antigen 3.2 Expression of AP205 cVLPs in E.coli 3.3 Purification of AP205 cVLPs 3.3.1 Lysis of Bacterial Cells 3.3.2 Purification by Ultracentrifugation (See Note 2) 3.4 Small-Scale Test Coupling of the Antigen to AP205 cVLPs 3.5 Vaccine Production 3.5.1 Endotoxin Removal from cVLPs 3.5.2 Final Vaccine Preparation 4 Notes References Chapter 10: Protein Nanocarriers Capable of Encapsulating Both Hydrophobic and Hydrophilic Drugs 1 Introduction 2 Materials 3 Methods 3.1 Fabrication of BSA Nanoparticles by Desolvation 3.2 Loading of Doxorubicin into BSA Nanoparticles 3.3 Loading of Bilirubin into BSA Nanoparticles 3.4 Purification of BSA Nanoparticles 3.5 Characterization of the z-Average Hydrodynamic Diameter of BSA Nanoparticles by Dynamic Light Scattering (DLS) Measurements 3.6 Characterization of the Morphology of BSA Nanoparticles by Scanning Electron Microscopy (SEM) 3.7 Construction of Calibration Curves for Doxorubicin 3.8 Construction of Calibration Curve for Bilirubin 3.9 Measurement of Drug Loading Capacity 4 Notes References Chapter 11: Unnatural Amino Acid Engineering for Intracellular Delivery of Protein Therapeutics 1 Introduction 1.1 Engineering EGFR-Targeted mCherry 1.2 Cancer Cell-Specific Delivery 1.3 Modular Conjugation of yCD Suicide Enzymes for Targeted Cancer Cell Therapy 2 Materials 2.1 Production of UAA-Modified mCherry and SpyTag- and SpyCatcher-Fusion Proteins 2.1.1 Culture Medium 2.1.2 Protein Expression and Cell Lysis 2.1.3 Protein Purification 2.2 Peptide Synthesis and Click Conjugation 2.3 SpyTag/SpyCatcher Conjugation 2.4 Protein and Protein Conjugate Analysis 2.4.1 SDS-PAGE 2.4.2 MALDI-TOF 2.5 Cell Culture and Protein Delivery 2.6 MTT Assay 2.7 Equipment 3 Methods 3.1 Production of UAA-Modified mCherry and SpyTag- and SpyCatcher-Fusion Proteins 3.1.1 Protein Expression and Cell Lysis 3.1.2 Protein Purification 3.2 Peptide Synthesis and Click Conjugation 3.2.1 Solid-Phase Peptide Synthesis 3.2.2 Peptide Purification 3.2.3 Click Conjugation 3.3 SpyTag/SpyCatcher Conjugation 3.3.1 Conjugation Reaction 3.4 Protein and Protein Conjugate Analysis 3.4.1 SDS-PAGE 3.4.2 MALDI-TOF 3.5 Protein Delivery Analyses 3.5.1 Mammalian Cell Culture 3.5.2 Protein Application 3.5.3 Flow Cytometry 3.6 Suicide Enzyme Activity Analyses 3.6.1 Protein and Prodrug Application 3.6.2 Cell Viability Analyses References Chapter 12: Formulation of Chitosan-Zein Nano-in-Microparticles for Oral DNA Delivery 1 Introduction 2 Materials 2.1 Materials to Form CS-DNA NPs 2.2 Materials to Encapsulate CS-DNA NPs into ZN 3 Methods 3.1 Preparation of CS-DNA-NPs 3.2 Encapsulate CS-DNA NPs in ZN to Make CS-ZN Nano-in-Microparticles 4 Notes References Chapter 13: Engineering E2 Bionanoparticles for Targeted Delivery of Chemotherapeutics to Breast Cancer Cells 1 Introduction 1.1 Engineering ST-E2-4F Nanocages for Targeted IBC Cell Delivery 1.2 Doxorubicin Encapsulation and Release in ST-E2-4F 1.3 Treatment of IBC Cells with DOX-Loaded 4GE11-E2-4F-ELP 2 Materials 2.1 Production of E2 Nanoparticles and SpyCatcher Fusion Proteins 2.1.1 Culture Medium 2.1.2 Protein Expression and Cell Lysis 2.2 Production of 4pAzF-mCherry-SpyCatcher 2.2.1 Culture Medium 2.2.2 Protein Expression and Cell Lysis 2.3 Synthesis of GE11 Peptide 2.3.1 Solid-Phase Synthesis 2.3.2 Cleavage from Resin 2.3.3 Equipment 2.4 Site-Specific Conjugation of GE11 Peptide to 4pAzF-mCherry Proteins 2.5 SpyTag/SpyCatcher Bioconjugation 2.5.1 SpyTag/SpyCatcher Reaction 2.6 Inverse Transition Cycling (ITC) for Purification of ELP Fusion Proteins 2.6.1 SDS-PAGE 2.6.2 Equipment 2.7 Cell Culture 2.8 Doxorubicin Encapsulation 2.9 E2 Nanoparticle Delivery 2.10 Flow Cytometry 2.10.1 Equipment 2.11 MTT Assay 3 Methods 3.1 Production of E2 Nanoparticles and SpyTag Fusion Proteins 3.1.1 Protein Expression and Cell Lysis 3.1.2 Purification of E2 Nanoparticles 3.1.3 Purification of SpyTag Fusion Proteins 3.2 SpyTag/SpyCatcher Conjugation 3.2.1 ELP Purification 3.2.2 SDS-PAGE 3.3 Expression of 4pAzF-mCherry-SpyCatcher 3.3.1 Purification of 4pAzF-mCherry-SpyCatcher 3.4 Mammalian Cell Culture 3.5 E2 Delivery and Flow Cytometry 4 MTT Viability Assay References Chapter 14: Constructing Nucleic Acid Delivering Lipoproteoplexes from Coiled-Coil Supercharged Protein and Cationic Liposomes 1 Introduction 2 Materials 2.1 Protein Expression 2.2 Protein Purification 2.3 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) 2.4 Dialysis and Protein Concentration 2.5 DOTAP:Na-Cholate (DNC) Liposome Preparation 2.6 Transfection 3 Methods 3.1 Protein Expression 3.2 Protein Purification 3.3 SDS-PAGE 3.4 Dialysis and Protein Concentration 3.5 DOTAP:Na-Cholate (DNC) Liposome Preparation 3.6 Transfection Protocol 4 Notes References Chapter 15: Cancer Cell Silicification and Surface Functionalization to Create Microbial Mimetic Cancer Vaccines 1 Introduction 2 Materials 2.1 Tumor Cell Preparation 2.2 Cell Cryo-Silicification 2.3 Surface Functionalization: Pathogen-Associated Molecular Patterns (PAMPs) 2.4 Silicified Cell Dehydration 3 Methods 3.1 Cancer Cell Enrichment from Ascites 3.2 Cryo-Silicification of Cancer Cells 3.3 Surface Functionalization of Silicified Cancer Cells 3.4 Dehydration and Rehydration of Vaccine Cells 4 Notes References Chapter 16: Melt Processing Virus-Like Particle-Based Vaccine Candidates into Biodegradable Polymer Implants 1 Introduction 2 Materials 2.1 Qβ VLP Expression and Purification 2.1.1 Instruments 2.1.2 Qβ VLP Expression 2.1.3 Qβ VLP Purification 2.2 HPV-Qβ Conjugation 2.3 HPV-Qβ Characterizations 2.3.1 SDS-Page 2.3.2 Native Agarose Gel Electrophoresis 2.3.3 Fast Protein Liquid Chromatography 2.3.4 Dynamic Light Scattering 2.3.5 Transmission Electron Microscopy 2.4 Preparation of Qβ/PLGA Implants by Hot-Melt Extrusion 2.5 Qβ/PLGA Implant Analysis 2.5.1 Scanning Electron Microscopy 2.5.2 In Vitro Release Kinetics 2.6 Animal Immunizations 2.7 Antibody Titer Determinations by ELISA 2.7.1 Equipment Needed for ELISA 2.7.2 HPV Peptide ELISA 2.7.3 Qβ ELISA 3 Methods 3.1 Qβ VLP Expression and Purification 3.2 HPV-Qβ Conjugation 3.3 HPV-Qβ Characterization 3.3.1 SDS-PAGE Is Used to Analyze Degree of Peptide Conjugation to Qβ Coat Protein 3.3.2 Particle Integrity Assessed by Native Agarose Gel Electrophoresis 3.3.3 Particle Purity and Integrity Assessed by Fast Protein Liquid Chromatography 3.3.4 Particle Size and Dispersity Measured by Dynamic Light Scattering 3.3.5 Visualization of Particle Integrity by Transmission Electron Microscopy 3.4 Preparation of VLP-Loaded PLGA Implants by Hot-Melt Extrusion 3.5 Implant Analysis 3.5.1 Scanning Electron Microscopy with Energy Dispersive X-Ray Spectroscopy (SEM-EDX) Is Used to Determine the Dispersion of ... 3.5.2 In Vitro Release Kinetics 3.6 Animal Immunization 3.7 HPV Peptide Antibody Titer Determination by ELISA 3.8 Carrier Antibody Titer Determination by ELISA 4 Notes References Index