Protein Cages: Design, Structure, and Applications (Methods in Molecular Biology, 2671)

Preface
Contents
Contributors
Part I: Design and Construction of Artificial Cages
	Chapter 1: Coiled-Coil Protein Origami: Design, Isolation, and Characterization
		1 Introduction
		2 Design and Modelling (CoCoPOD)
			2.1 CoCoPOD Installation (See Note 1)
			2.2 Polyhedral Designs with CoCoPOD (See Note 4)
		3 Cloning
			3.1 Materials
				3.1.1 Golden Gate Assembly
				3.1.2 Colony PCR and Screening
			3.2 Methods
				3.2.1 Golden  Gate
				3.2.2 Colony PCR and Screening
		4 Fermentation and Isolation
			4.1 Materials
				4.1.1 Bacterial Fermentation for Production of Proteins
				4.1.2 Lysis of Bacterial Cells
				4.1.3 Ultrasonication
				4.1.4 Thermal Lysis
				4.1.5 NiNTA Affinity Chromatography
				4.1.6 Strep-Tag Affinity Chromatography (See Note 36)
				4.1.7 Ion-Exchange Chromatography (See Note 36)
				4.1.8 Size Exclusion Chromatography (See Note 36)
				4.1.9 TEV Protease Cleavage
				4.1.10 Reverse NiNTA
			4.2 Methods
				4.2.1 Bacterial Fermentation for Production of Proteins
				4.2.2 Lysis of Bacterial Cells
				4.2.3 Ultrasonication
				4.2.4 Thermal Lysis
				4.2.5 NiNTA Affinity Chromatography
				4.2.6 Strep-Tag Affinity Chromatography
				4.2.7 Ion-Exchange Chromatography
				4.2.8 Size Exclusion Chromatography
				4.2.9 TEV Protease Cleavage and Reverse NiNTA (See Note 102)
		5 Characterization
			5.1 Materials
				5.1.1 Circular Dichroism Spectroscopy
				5.1.2 Size Exclusion Chromatography Coupled with Multiangle Light Scattering (SEC-MALS) (See Note 36)
				5.1.3 Small-Angle X-Ray Scattering (SAXS)
			5.2 Methods
				5.2.1 Circular Dichroism Spectroscopy
				5.2.2 Size Exclusion Chromatography Coupled with Multiangle Light Scattering (SEC-MALS)
				5.2.3 Small-Angle X-Ray Scattering (SAXS)
		6 Notes
		References
	Chapter 2: Artificial Protein Cages Assembled via Gold Coordination
		1 Introduction
		2 Materials
			2.1 TRAP Protein
			2.2 TRAP K35C/R64S Expression Plasmid
			2.3 Protein Expression
			2.4 Protein Purification
			2.5 Tris-Tricine SDS-PAGE
			2.6 Chromatography Columns
			2.7 Source of Metal  Ions
			2.8 TRAP-Cage Self-assembly Reaction and Purification
			2.9 TRAP-Cage Validation and Characterization
			2.10 Native  PAGE
			2.11 TRAP-Cage Cellular Toxicity Tests
			2.12 Transformed Cells for Protein Expression
		3 Methods
			3.1 TRAP Protein Expression and Purification
			3.2 Tris-Tricine SDS-PAGE
			3.3 TRAP-Cage Self-assembly Reaction
			3.4 TRAP-Cage Validation and Characterization
				3.4.1 TRAP-Cage Stability Tests
				3.4.2 Dark Blue Native  PAGE
				3.4.3 Transmission Electron Microscopy (TEM)
				3.4.4 Dynamic Light Scattering (DLS) Measurement
			3.5 TRAP-Cage Cellular Toxicity Tests
				3.5.1 Resazurin (Alamar Blue) Assay
			3.6 Further Characterization
		4 Notes
		References
	Chapter 3: Reassembly Design of Ferritin Cages
		1 Introduction
		2 Materials
			2.1 Protein Interface Redesign
			2.2 Protein Expression and Purification
			2.3 Protein Characterization
		3 Methods
			3.1 Protein Interface Redesign
			3.2 Protein Preparation
			3.3 Protein Characterization
			3.4 Protein Crystallization, Data Collection, and Structure Determination
		4 Notes
		References
	Chapter 4: Protein Cages and Nanostructures Constructed from Protein Nanobuilding Blocks
		1 Introduction
		2 Materials
			2.1 DNA Construction
			2.2 Protein Expression and Purification
			2.3 Equipment and Facility
		3 Methods
			3.1 Construction of Polyhedral Protein Nanobuilding Block (PN-Block)
				3.1.1 Design of Polyhedral PN-Block (WA20-Foldon)
				3.1.2 Construction of Expression Plasmid of WA20-Foldon
				3.1.3 Protein Expression of WA20-Foldon
				3.1.4 Purification of WA20-Foldon
			3.2 Construction of Extender Protein Nanobuilding Blocks (ePN-Blocks)
				3.2.1 Design of ePN-Blocks
				3.2.2 Construction of Protein Expression Plasmids of ePN-Blocks
				3.2.3 Protein Expression of ePN-Blocks
				3.2.4 Purification of ePN-Blocks
			3.3 Reconstruction of Heterooligomeric Complexes of ePN-Block and sPN-Block
				3.3.1 Denaturation and Refolding of ePN-Block and sPN-Block
				3.3.2 Purification of esPN-Block Complexes
			3.4 Analyses of PN-Blocks
				3.4.1 Polyacrylamide Gel Electrophoresis (PAGE)
				3.4.2 Size Exclusion Chromatography-Multi-Angle Light Scattering (SEC-MALS) Analysis
				3.4.3 Small-Angle X-Ray Scattering (SAXS) Analysis
				3.4.4 Low-Resolution Shape Modeling of PN-Block Complexes Based on SAXS Analysis
				3.4.5 Rigid-Body Modeling of PN-Block Complexes Based on SAXS Analysis
		4 Notes
		References
	Chapter 5: Preparation of Cage-Like Micellar Assemblies of Engineered Hemoproteins
		1 Introduction
		2 Materials
			2.1 Expression and Purification of Cytochrome b562 Mutant
			2.2 Expression and Purification of HTHP Mutant
			2.3 Modification of Cytochrome b562 Mutant by Synthetic Heme
			2.4 Modification of HTHP with Maleimide-Tethering PNIPAAm
			2.5 Characterization of Cage-like Micellar Structures
		3 Methods
			3.1 Design of Protein Mutants
			3.2 Expression and Purification of Cytochrome b562 Mutant
			3.3 Expression and Purification of HTHP Mutant
			3.4 Modification of Cytochrome b562 Mutant by Synthetic Heme
			3.5 Modification of HTHP with Maleimide-Tethering PNIPAAm
			3.6 Characterization of Cage-Like Micellar Structures
				3.6.1 Sample Preparation for Dynamic Light Scattering
				3.6.2 Sample Preparation for Transmission Electron Microscopy
				3.6.3 Sample Preparation for Atomic Force Microscopy
		4 Notes
		References
Part II: Functionalization of Protein Cages
	Chapter 6: Fabrication of Protein Macromolecular Frameworks (PMFs) and Their Application in Catalytic Materials
		1 Introduction
		2 Materials
			2.1 Preparation of VLP Building Blocks
			2.2 Construction of PMFs
			2.3 Activity Assay of AdhD-Enclosed PMF with Charged Substrates
		3 Methods
			3.1 Transformation of the P22 VLP Morphology from PC to EX  Form
			3.2 Transformation of the P22 VLP Morphology from PC to WB  Form
			3.3 Preparation of Ditopic Protein Linker from DecS134C
			3.4 Construction of PMFs from the EX or WB Form of P22 VLP
			3.5 Activity Assay of AdhD-Enclosed PMF with Charged Substrates
		4 Notes
		References
	Chapter 7: Iron Accumulation in Ferritin
		1 Introduction
		2 Materials
			2.1 Preparation of Ferritin and Fe2+ Solution for In Vitro Iron Mineralization
			2.2 Preparation of Non-denaturing Polyacrylamide Gel for Checking Iron Accumulation in Ferritin Cage
			2.3 Preparation of Ferritin-Mount Grid for Transmission Electron Microscopy (TEM)
			2.4 Quantification of Accumulated Iron inside Ferritin Nanocage
			2.5 Equipment and Software
			2.6 Other Consumables
		3 Methods
			3.1 In Vitro Iron Mineralization in Ferritin
			3.2 Checking Iron Accumulation in Ferritin Cage by Native-PAGE
			3.3 Estimation of Size of Ferritin Iron Mineral Core and its Shell by Transmission Electron Microscopy (TEM)
			3.4 Quantification of Accumulated Iron inside the Ferritin Nanocage
		4 Notes
		References
	Chapter 8: A Generalized Method for Metal Fixation in Horse Spleen L-Ferritin Cage
		1 Introduction
		2 Materials
			2.1 Preparation of Apo-Recombinant Horse Spleen L-Ferritin
			2.2 Metal Fixation
			2.3 Crystallization
			2.4 Purification and Characterization
		3 Methods
			3.1 Purification of Apo-rHLFr
			3.2 Immobilization of Synthetic Metal Complexes into Apo-rHLFr
			3.3 Purification of the Apo-rHLFr Composites
			3.4 Immobilization of Metal Ions into Apo-rHLFr
			3.5 Crystallization of the Apo-rHLFr Composites
		4 Notes
		References
	Chapter 9: Dual Modification of Artificial Protein Cage
		1 Introduction
		2 Materials
			2.1 Proteins and Materials for Their Purifications
			2.2 Chemicals for Modification Reactions
		3 Methods
			3.1 Preparation of Three Mutants
			3.2 Optimization of the Reaction Conditions for Estimation of Pore Size of TIP60
			3.3 Estimation of the Actual Pore Size of Protein Nanocages in Solution
			3.4 Dual-Surface Modifications
			3.5 Release of Bound TNB to Cys Residue on the Interior Surface of TIP60
		4 Notes
		References
	Chapter 10: Modification and Production of Encapsulin
		1 Introduction
		2 Materials
			2.1 Cloning
			2.2 Production
			2.3 Purification Steps
			2.4 Characterization
		3 Methods
			3.1 Cloning Steps
			3.2 Production Steps
			3.3 Purification Steps
			3.4 Characterization
		4 Remarks and Conclusion
		5 Notes
		References
Part III: Characterization and Simulation of Protein Cage Systems
	Chapter 11: Cryo-electron Microscopy of Protein Cages
		1 Introduction
		2 Materials
			2.1 Cryo-EM Data Collection
			2.2 Image Processing
		3 Methods
			3.1 Sample Preparation
			3.2 Grid Selection, Preparation, and Sample Application
			3.3 Data Acquisition
				3.3.1 Physical Hardware
				3.3.2 Accelerating Voltage
				3.3.3 Magnification
				3.3.4 Electron Dose
				3.3.5 Defocus
				3.3.6 Quantity of  Data
			3.4 Image Processing
				3.4.1 Importing Micrograph Movies
				3.4.2 Motion Correction
				3.4.3 Contrast Transfer Function Estimation
				3.4.4 Particle Selection
				3.4.5 Particle Classification
				3.4.6 Initial Model Generation
				3.4.7 3D Reconstruction and Refinement
				3.4.8 Analysis and Validation of Cryo-EM  Maps
				3.4.9 Special Considerations for Giant Particles
			3.5 Summary and Outlook
			3.6 Data Availability
		4 Notes
		References
	Chapter 12: Time-Resolved Small-Angle X-Ray Scattering of Protein Cage Assembly
		1 Introduction
		2 Materials
			2.1 Preparation of Solutions
			2.2 Instruments (Beamline Setup)
		3 Methods
			3.1 Experimental Procedures
			3.2 Data Processing
		4 Notes
		References
	13: Protein Cage-Stabilized Emulsions: Formulation and Characterization
		1 Introduction
		2 Materials
			2.1 Pickering Emulsion Formulation
			2.2 Dynamic Light Scattering (DLS)
			2.3 Tryptophan Fluorescence (TF) Intensity
			2.4 Circular Dichroism (CD)
			2.5 Small Angle X-Ray Scattering (SAXS)
		3 Methods
			3.1 Emulsion Formulation
			3.2 Droplet Size Measurement by DLS
			3.3 Tertiary Structure Analysis by TF Intensity
			3.4 Secondary Structure Analysis by Far-UV (CD)
			3.5 Protein Cage in Bulk and at the Interface Structure Determination using SAXS
		4 Notes
		References
	14: A Method to Investigate the Mechanism of Charge Transport Across Bio-Molecular Junctions with Ferritin
		1 Introduction
		2 Materials
			2.1 Equipment
		3 Methods
			3.1 Fabrication of Template Stripped Au Bottom-Electrodes
			3.2 Template-Stripped Nickel (NiTS)
			3.3 Self-Assembly of Protein Monolayer on the Bottom Electrode
			3.4 Fabrication of EGaIn Electrodes Stabilized in a Through-Hole
			3.5 Fabrication of Micropore-Based Junction
			3.6 Electrical Characterization
				3.6.1 Data Collection of DC Measurements
				3.6.2 Temperature-Dependent Measurements
		4 Notes
		References
	15: Potato Virus X Inactivation and Characterization
		1 Introduction
		2 Materials
			2.1 PVX Inactivation
			2.2 Fluorophore Conjugation
			2.3 Virus Particle Characterization
			2.4 In Planta Inoculation
			2.5 Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
		3 Methods
			3.1 PVX Inactivation
				3.1.1 UV Inactivation
				3.1.2 βPL Inactivation
				3.1.3 Formalin Inactivation
			3.2 Oregon Green 488 (O488) Conjugation to PVX
				3.2.1 Thiol-Maleimide Chemistry to Conjugate O488 to PVX´s Cysteines
				3.2.2 N-Hydroxysuccinimide (NHS)-Amine Chemistry to Conjugate O488 to PVX´s Lysines
			3.3 Particle Characterization
				3.3.1 UV-Vis Spectroscopy
				3.3.2 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS PAGE)
				3.3.3 Dynamic Light Scattering (DLS)
				3.3.4 Size Exclusion Chromatography (SEC)
				3.3.5 Transmission Electron Microscopy (TEM)
				3.3.6 PVX RNA Extraction
				3.3.7 RNA Agarose Gel Electrophoresis
			3.4 PVX Plan Infectivity
				3.4.1 Plant Inoculation
				3.4.2 RNA Extraction from Leaves
				3.4.3 RT-PCR
				3.4.4 DNA Agarose Gel Electrophoresis
		4 Notes
		References
	16: Molecular Dynamics Simulation of Protein Cages
		1 Introduction
		2 MD Simulation Workflow
			2.1 System Preparation
				2.1.1 Initial Structure
				2.1.2 Protonation States
				2.1.3 Topology Generation
				2.1.4 Solvation
				2.1.5 Neutralization
			2.2 Equilibration
				2.2.1 Energy Minimization
				2.2.2 Constrained Pre-equilibration
				2.2.3 Equilibration Without Position Constraint
			2.3 Production Simulation
			2.4 Analysis of MD Trajectories
				2.4.1 RMSD
				2.4.2 RMSF
				2.4.3 Radius of Gyration
				2.4.4 Radial Distribution Function
				2.4.5 Custom Physical Properties
		3 MD Simulation with GROMACS
			3.1 Preparation of the System
				3.1.1 Initial Structure
				3.1.2 Protonation States
				3.1.3 Topology Generation
				3.1.4 Solvation
				3.1.5 Neutralization
			3.2 Equilibration
				3.2.1 Energy Minimization
				3.2.2 Constrained Pre-equilibration
				3.2.3 Equilibration Without Position Constraint
			3.3 Production Simulation
			3.4 Analysis of MD Trajectories
				3.4.1 RMSD
				3.4.2 RMSF
				3.4.3 Radius of Gyration
				3.4.4 Radial Distribution Function
				3.4.5 Custom Physical Properties
		4 MD Simulation with NAMD
			4.1 Preparation of the System
			4.2 Equilibration
				4.2.1 Energy Minimization
				4.2.2 Constrained Pre-equilibration
				4.2.3 Equilibration Without Position Constraint
			4.3 Production Simulation
		References
	Chapter 17: Coarse-Grained Models for Vault Normal Model Analysis
		1 Introduction
		2 Materials
		3 Methods
			3.1 Multiscale Virtual Particle-Based Anisotropic Network Model (MVP-ANM)
				3.1.1 Multiscale Representation of Biomolecules
				3.1.2 Generalized Spring Parameter
				3.1.3 Multiscale Virtual Particle-Based Anisotropic Network Model (MVP-ANM)
			3.2 Vault Dynamics Analysis with MVP-ANM
		4 Notes
		References
Part IV: Translation of Protein Cages
	18: Evaluating Anti-tumor Immune Responses of Protein Nanoparticle-Based Cancer Vaccines
		1 Introduction
		2 Materials
			2.1 Antigen and Adjuvant Conjugation in D381C (E2)
			2.2 Cytotoxic T Lymphocyte (CTL) Lysis Assay
			2.3 Interferon-Gamma (IFN-γ) ELISpot
			2.4 Tumor Challenge
		3 Methods
			3.1 Antigen and Adjuvant Conjugation to E2 Protein Nanoparticle
			3.2 CTL Lysis Assay
			3.3 IFN-γ ELISpot
			3.4 Tumor Challenge
		4 Notes
			4.1 Antigen and Adjuvant Conjugation
			4.2 CTL Assay
			4.3 IFN-γ ELISpot
			4.4 Tumor Challenge
		References
	19: Construction Protocol of Drug-Protein Cage Complexes for Drug Delivery System
		1 Introduction
		2 Materials
			2.1 Ferritin Production
			2.2 Protein Analysis
			2.3 Drug-Ferritin Complex Construction
			2.4 Equipment
		3 Methods
			3.1 Plasmid Construction
			3.2 Protein Production
			3.3 Disassembly/Reassembly Method
			3.4 One-Step Method
			3.5 Evaluation of Drug-Protein Complex
		4 Notes
		References
	20: Protein Cage Relaxivity Measurement for Magnetic Resonance Imaging Contrast Agents
		1 Introduction
		2 Materials
			2.1 Consumables
			2.2 Protein Cage Loaded with Metal  Ions
			2.3 Chemicals
			2.4 Buffer
			2.5 Equipment
		3 Methods
			3.1 Iron Loading
			3.2 Concentration Estimation of Ferritin Cage Bound Iron Using ICP-MS
			3.3 Sample Preparation for Relaxivity Measurement
			3.4 Water Proton Relaxation Time Measurements using NMR
				3.4.1 T1 Relaxation
				3.4.2 T2 Relaxation
				3.4.3 Calculation of T1 and T2 Values
			3.5 Water Proton Relaxation Time Measurement Using MRI
				3.5.1 T1 and T2 Pulse Sequences Used for the Generation of MRI  Data
				3.5.2 Protocol for T1 and T2 Relaxation Measurements
				3.5.3 Data Processing
			3.6 Relaxivity r1 and r2 Calculation from T1 and T2 Values
		4 Notes
		References
	21: Crystalline Biohybrid Materials Based on Protein Cages
		1 Introduction
		2 Materials
			2.1 Materials for Computational Design of Supercharged Ferritin Variants
			2.2 Materials for Protein Production and Purification of Supercharged Ferritin Variants
				2.2.1 Cell Culture
				2.2.2 Protein Purification
			2.3 Materials for Synthesis of Metal Oxide Nanoparticles Inside the Ferritin Cavity
				2.3.1 Iron Oxide Nanoparticles
				2.3.2 Cobalt Oxide Nanoparticles
				2.3.3 Cerium Oxide Nanoparticles
			2.4 Materials for Protein Crystallization
		3 Methods
			3.1 Methods for Computational Design of Supercharged Ferritin Variants
				3.1.1 Preparing the Input
				3.1.2 Generate a Symmetry Definition  File
				3.1.3 Energy Minimization of the Input Structure
				3.1.4 Identification of Possible Mutation Sites
			3.2 Methods for Protein Production and Purification of Supercharged Ferritin Variants
				3.2.1 Protein Production of Ftn(pos)
				3.2.2 Protein Production of Ftn(neg)
				3.2.3 Protein Purification
					Purification of Ftn(pos)
					Purification of Ftn(neg)
					Size Exclusion Chromatography
			3.3 Methods for Synthesis of Metal Oxide Nanoparticles Inside the Ferritin Cavity
				3.3.1 Iron Oxide Nanoparticle Synthesis
				3.3.2 Cobalt Oxide Nanoparticle Synthesis
				3.3.3 Cerium Oxide Nanoparticle Synthesis
				3.3.4 Gel Filtration for Nanoparticle Loaded Protein Cages
				3.3.5 Sucrose Gradient Centrifugation of Nanoparticle-Loaded Samples
			3.4 Methods for Protein Crystallization
			3.5 Methods for Small Angle X-Ray Scattering (SAXS) Measurement
				3.5.1 Protein Crystal Fixation
				3.5.2 SAXS Measurement
					Data Collection
					Data Analysis
		4 Notes
		References
	22: Production and Purification of Virus-Like Particles by Transient Expression in Plants
		1 Introduction
		2 Materials
			2.1 Lab Equipment
			2.2 Reagents and Consumables
		3 Methods
			3.1 Plant Growth
			3.2 Infiltration
			3.3 Purification I: Harvest, Lysis and Clarification, Differential Centrifugation
				3.3.1 Harvesting
				3.3.2 Tissue Disruption and Clarification
				3.3.3 Differential Centrifugation
				3.3.4 Desalting
			3.4 Purification II: Size Exclusion Chromatography and Dynamic Light Scattering
				3.4.1 SEC
				3.4.2 DLS
			3.5 Compositional Analysis
				3.5.1 Concentration Determination by Bradford Assay
				3.5.2 SDS-PAGE
				3.5.3 TEM
		4 Notes
		References
	23: Laboratory Scale Production of Complex Proteins Using Charge Complimentary Nanoenvironments
		1 Introduction
		2 Materials
			2.1 Bacterial Expression
			2.2 Isolation of Proteins
			2.3 Purification of Proteins
			2.4 Protein Folding
		3 Methods
			3.1 Preparation of tES Variant Clones
			3.2 Preparation of h6POI (6xHistidine-Protein-of-Interest) Clones
			3.3 Expression of tES Variants
			3.4 Expression of h6POI
			3.5 Isolation and Purification of tES Variants
			3.6 Isolation and Purification of h6POIs
			3.7 In Vitro Folding
		4 Notes
		References
Index