Recombinant Proteins in Plants: Methods and Protocols (Methods in Molecular Biology, 2480)

✍ Scribed by Stefan Schillberg (editor), Holger Spiegel (editor)

• Publisher: Humana
• Year: 2022
• Tongue: English
• Leaves: 345
• Category: Library

✦ Synopsis

This volume provided methods and protocols on recombinant protein production in different plant systems, downstream processing, and strategies to optimize protein expression. Chapters guide readers through recombinant protein production in important plant systems, protein recovery and purification, different strategies to optimise productivity, cloning and fusion protein approaches, and the regulation and freedom to operate analysis of plant-produced proteins. 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.

Authoritative and cutting-edge, Recombinant Proteins in Plants: Methods and Protocols aims to be useful to newcomers and experienced researchers interested in expanding their expertise in the field of plant-based protein production.

Chapters 6, 8 and 17 are available open access under a Creative Commons Attribution 4.0 International License via link.springer.com.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Recombinant Protein Production in Plants: A Brief Overview of Strengths and Challenges
1 Why Are Plants Used for Protein Production?
2 Which Plants Are Used for Protein Production?
3 What Challenges Remain to Be Addressed?
4 Notes
References
Part I: Plant Production Systems and Applications
Chapter 2: Production of Recombinant Proteins in Transgenic Tobacco Plants
1 Introduction
2 Materials
2.1 Freeze-Thaw Method for Agrobacterium tumefaciens Transformation
2.2 Agrobacterium-Mediated Transformation and In Vitro Regeneration of Plantlets
2.3 Transfer of In Vitro-Grown Plantlets to Soil, Cross-Fertilization, Seed Collection
2.4 ELISA
2.5 SDS Polyacrylamide Gel Electrophoresis
2.6 Semi-Dry Enhanced Chemiluminescence (ECL) Western Blot
2.7 Purification of Recombinant Protein from Tobacco by Affinity Chromatography
2.8 Surface Plasmon Resonance
3 Methods
3.1 Freeze-Thaw Method for Agrobacterium tumefaciens Transformation
3.2 Agrobacterium-Mediated Transformation of and In Vitro Regeneration of Plantlets
3.3 Transfer of In Vitro-Grown Plantlets to Soil, Cross-Fertilization, Seed Collection
3.4 ELISA
3.4.1 Direct ELISA for Measuring the Presence of α(1,3)-Fucose
3.4.2 Indirect ELISA for Characterizing HIV Antigen Binding Capacity
3.4.3 Sandwich ELISA for Determining VRC01 Concentration
3.5 SDS Polyacrylamide Gel Electrophoresis
3.6 Semi-Dry Enhanced Chemiluminescence (ECL) Western Blot
3.7 Purification of Recombinant Protein from Tobacco by Affinity Chromatography
3.8 Surface Plasmon Resonance
3.8.1 Immobilization of Protein A onto CM5 Chip for Direct and Capture Assays
3.8.2 Direct Assay for Measuring Concentration of Purified VRC01
3.8.3 Capture Assay for Measuring Binding Kinetics of Glycoengineered VRC01
4 Notes
References
Chapter 3: Molecular Farming in Seed Crops: Gene Transfer into Barley (Hordeum vulgare) and Wheat (Triticum aestivum)
1 Introduction
2 Material
2.1 Plant Donor Material
2.2 Biolistic Genetic Transformation of Immature Barley and Wheat Embryos
2.2.1 Seed Surface Sterilization
2.2.2 Immature Embryo Isolation
2.2.3 DNA Transfer Using the Biolistic PDS-1000/He Unit
2.3 Barley Tissue Culture Media
2.4 Wheat Tissue Culture Media
2.5 Plant Genomic DNA Isolation
2.6 PCR
3 Methods
3.1 Growth Conditions for Donor Plant Material (See Note 6)
3.2 Biolistic Transformation of Immature Barley and Wheat Embryos
3.2.1 Seed Surface Sterilization
3.2.2 Immature Embryo Isolation
3.2.3 DNA Transfer Using the Biolistic PDS-1000/He Unit
3.3 Cultivation of Barley Explants
3.4 Cultivation of Wheat Explants
3.5 Genomic DNA Isolation
3.6 Detection of the Gene-of-Interest by PCR
4 Notes
References
Chapter 4: Cell Biology Methods to Study Recombinant Proteins in Seeds
1 Introduction
2 Material
2.1 Sequential Extraction of Recombinant Proteins from Seeds
2.2 Subcellular Fractionation on Isopycnic Sucrose Gradients
2.3 Isolation of Maize (Zea mays) Protein Bodies by Discontinuous Sucrose Gradient Centrifugation
2.4 3D Electron Microscopy: Serial Block Face Imaging and Electron Tomography
3 Methods
3.1 Sequential Extraction of Recombinant Proteins
3.2 Subcellular Fractionation on Isopycnic Sucrose Gradients (See Note 15)
3.3 Isolation of Maize PBs by Discontinuous Sucrose Gradient Centrifugation (See Note 38)
3.4 3D Electron Microscopy: Serial Block Face Imaging and Electron Tomography (See Note 43)
3.4.1 Sample Preparation
3.4.2 Serial Block Face Imaging
3.4.3 Electron Tomography
4 Notes
References
Chapter 5: Production of Recombinant Glycoproteins in Nicotiana tabacum BY-2 Suspension Cells
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipments
3 Methods
3.1 Obtaining an Agrobacterium tumefaciens with the Binary Plasmid of Interest
3.2 Maintenance of Nicotiana tabacum BY-2 Cell Culture
3.3 Growth of Agrobacterium tumefaciens and Preparation of the Inoculum
3.4 Co-cultivation of Nicotiana tabacum BY-2 Cells with Agrobacterium tumefaciens
3.5 Selection of Transgenic BY-2 Cell Lines
3.6 Selection of BY-2 Elite Lines
4 Notes
References
Chapter 6: Production of Recombinant Proteins by Agrobacterium-Mediated Transient Expression
1 Introduction
2 Materials
2.1 Construct Cloning and Sequencing
2.2 Agrobacterium Cultivation
2.3 Plant Cultivation and Infiltration
2.4 Protein Extraction
3 Methods
3.1 Preparation of Expression Constructs
3.2 Identification of Recombinant E. coli Cells
3.3 Preparation of Electrocompetent A. tumefaciens Cells
3.4 Electroporation of A. tumefaciens
3.5 Identification of Recombinant A. tumefaciens Cells
3.6 Cultivation of Recombinant A. tumefaciens Cells
3.7 Preparation of the Infiltration Solution
3.8 Cultivation of N. benthamiana Plants
3.9 Plant Infiltration and Incubation
3.9.1 Syringe-Based Infiltration (A)
3.9.2 Vacuum Infiltration (B)
3.10 Extraction of Total Soluble Protein (TSP)
4 Notes
References
Chapter 7: Specific Packaging of Custom RNA Molecules into Cowpea Mosaic Virus-like Particles
1 Introduction
2 Materials
2.1 Media, Buffers, Solutions
2.2 Equipment
2.3 Enzymes
2.4 Plasmids
2.5 Bacterial Strains
2.6 Plants
3 Methods
3.1 Creation of Expression Plasmid Containing Custom Sequence of Choice
3.1.1 Choice of Expression Vector
3.1.2 Restriction Enzyme-Based Cloning
3.2 Transformation of Agrobacterium
3.3 Agroinfiltration of N. benthamiana
3.3.1 Preparation of Agrobacterium Suspensions
3.3.2 Infiltration of Leaves with Agrobacterium Suspensions
3.4 Extraction and Purification
4 Notes
References
Chapter 8: Plant-Based Cell-Free Transcription and Translation of Recombinant Proteins
1 Introduction
2 Materials
2.1 Preparation of the Expression Vector
2.2 Coupled Transcription-Translation Reaction
2.3 Protein Recovery and Affinity Purification
3 Methods
3.1 Preparation of the Expression Vector
3.2 Coupled Transcription-Translation Reaction
3.2.1 General Procedures
3.2.2 Coupled Transcription-Translation Reaction: 50-μL Scale in Microtiter Plates
3.2.3 Coupled Transcription-Translation Reaction: 52-μL Scale in 2-mL Tubes
3.2.4 Coupled Transcription-Translation Reaction at Volumes of 4-6 mL in 50-mL Bioreactor Tubes
3.3 Protein Recovery and Affinity Purification
3.3.1 Recovery of Recombinant Proteins from the Microsomes
3.3.2 Purification of Strep-II-Tagged Proteins by Affinity Chromatography
4 Notes
References
Part II: Downstream Processing
Chapter 9: Strategies for Efficient and Sustainable Protein Extraction and Purification from Plant Tissues
Abbreviations
1 Introduction
2 Materials
2.1 Heat Precipitation (Optional)
2.2 Extraction and Filtration
2.3 Enhanced Clarification and Conditioning (Optional)
2.4 Chromatography
3 Methods
3.1 Heat Precipitation (Optional)
3.2 Extraction and Clarification
3.2.1 Extraction Using a Blender
3.2.2 Extraction Using a Screw Press (Alternative)
3.3 Enhanced Clarification and Conditioning
3.3.1 Flocculation of Dispersed Particles in Plant Extracts (Optional)
3.3.2 Filter Additives (Conditional)
3.3.3 Ultrafiltration/Diafiltration (Optional)
3.4 Chromatography
4 Notes
References
Chapter 10: Improving Recombinant Protein Recovery from Plant Tissue Using Heat Precipitation
1 Introduction
2 Materials
2.1 Buffers, Reagents and Consumables
2.2 Equipment
3 Methods
3.1 Extraction of Total Soluble Proteins
3.2 Heat Precipitation
3.3 Blanching of Intact Plant Tissues
3.4 Sample Analysis
3.5 Scale-up Considerations
4 Notes
References
Chapter 11: Technoeconomic Modeling and Simulation for Plant-Based Manufacturing of Recombinant Proteins
1 Introduction
2 Materials
2.1 Technical Considerations of SuperPro Designer
2.2 Plant-Based Manufacturing Limitations with SuperPro Designer
2.3 Technoeconomic and Plant-Based Manufacturing Resources
3 Method
3.1 Process Creation
3.1.1 Stop Gate I
3.2 Process Synthesis
3.2.1 Mass and Energy Balances
3.2.2 Labor and Scheduling
3.2.3 Equipment, Consumables, and Utilities
3.2.4 Branches and Sections
3.2.5 Economics
3.2.6 Environmental Impact
Environmental, Health, and Safety Assessment
Process Mass Index (PMI)
3.2.7 Stop Gate II
3.3 Process Analysis
3.3.1 Price Sensitivity
3.3.2 Scenario Analysis
3.3.3 Alternate Scenarios
3.3.4 Stop Gate III
4 Notes
References
Part III: Optimization Strategies
Chapter 12: Optimization of Vectors and Targeting Strategies Including GoldenBraid and Genome Editing Tools: GoldenBraid Assem...
1 Introduction
2 Materials
2.1 GoldenBraid Plasmids
2.2 Software Tools
2.3 GB Cloning
2.4 Bacteria Transformation and Culture
2.5 Plant Transient Transformation
2.6 DNA Extraction and PCR Amplification
3 Methods
3.1 Identification of Target Gene(s) of Interest for Editing
3.2 Considerations for the Selection of RNA Guides for LbCas12a
3.3 Design of Cas12a Multiplexed crRNA Guides with GoldenBraid
3.4 Cloning the (Protospacer-Scaffold)n in pUPD2 to Generate a Level 0 Part
3.5 Level 1 Polycistronic crRNA Guide Expression Cassette Assembly
3.6 Final T-DNA Expression Vector Assembly (Level 2 Part)
3.7 Transient Expression in N. benthamiana Leaves
3.8 Functional Validation of the Generated Construct for Multiplexing Gene Editing
4 Notes
References
Chapter 13: Advanced Fusion Strategies for the Production of Functionalized Potato Virus X Virions
1 Introduction
2 Materials
2.1 Genetic Engineering
2.2 PVX Particle Production
2.3 Plant Virus Purification
2.4 Protein Expression
2.4.1 Transient Expression in Plants
2.4.2 Expression in E. coli
2.5 Protein Purification
2.6 Protein and Particle Analysis
2.6.1 Imaging
2.6.2 RNA Analysis
2.6.3 SDS-PAGE/Western Blotting
2.7 SpyTag/SpyCatcher Coupling
2.8 Transmission Electron Microscopy (TEM)
2.8.1 Adsorption Grids
2.8.2 Immunosorbent TEM
3 Methods
3.1 Genetic Engineering
3.1.1 Construction of PVX Clones
3.1.2 Construction of SpyCatcher Fusion Proteins
3.2 PVX Particle Production
3.3 Plant Virus Purification
3.4 Protein Expression
3.4.1 Transient Expression in Plants
3.4.2 Expression in E. coli
3.5 Protein Purification
3.5.1 Purification of Proteins Expressed in Plants
3.5.2 Purification of Proteins Expressed in E. coli
3.6 Protein and Particle Analysis
3.6.1 Visualization of Fluorescence (See Fig. 2)
3.6.2 RNA Analysis
3.6.3 Gel Electrophoresis and Immunoblotting (Fig. 3)
3.7 SpyTag/SpyCatcher Coupling Reactions
3.8 Density of Fusion Proteins Presented on the Particle Surface
3.9 Transmission Electron Microscopy
3.9.1 Adsorption Grids
3.9.2 Immunosorbent Transmission Electron Microscopy (ISEM)
4 Notes
References
Chapter 14: Knockout of Glycosyltransferases in Nicotiana benthamiana by Genome Editing to Improve Glycosylation of Plant-Prod...
1 Introduction
1.1 Gene Knockout with CRISPR/Cas9
1.1.1 Multiplex Gene Knockout
1.2 Outline of the Workflow
2 Materials
2.1 Target Gene Resequencing
2.1.1 Spin-Column Extraction of Plant Genomic DNA
2.1.2 Polymerase Chain Reaction of Genomic DNA
2.1.3 Agarose Gel Electrophoresis
2.1.4 Spin-Column Purification of Linear DNA Fragments
2.1.5 Sanger Sequencing
2.1.6 Analysis of Sanger Sequencing Results
2.2 gRNA Design, Cloning and Testing
2.2.1 Cloning of gRNA Test Constructs
2.2.2 Transformation of Chemically Competent E. coli
2.2.3 Colony PCR
2.2.4 Liquid Cultivation of E. coli
2.2.5 Plasmid Preparation from E. coli
2.2.6 Preparation of Electrocompetent Agrobacterium tumefaciens Cells for Electroporation
2.2.7 Transformation of Electrocompetent A. tumefaciens
2.2.8 Colony PCR of Electroporated A. tumefaciens
2.2.9 Liquid Cultivation of A. tumefaciens
2.2.10 Agroinfiltration
2.2.11 Analysis of gRNA Efficiency
2.3 Stable Transformation of Plants
2.3.1 Cloning of the Transformation Construct
2.3.2 Agrotransformation and Agroinfiltration
2.3.3 Plant Transformation and Regeneration
2.4 Genetic Analysis of Regenerated Plants
2.4.1 Fast and Easy Extraction of Plant Genomic DNA
2.4.2 Target Gene Amplification
2.4.3 Clean-Up of PCR Products
2.4.4 Sanger Sequencing of Purified PCR Products
2.5 Immunoblot Analysis of Regenerated Plants
2.5.1 Dot Blot Analysis
2.5.2 Protein Extraction for SDS-PAGE
2.5.3 SDS-PAGE
2.5.4 Microwave-Assisted Coomassie Staining of Acrylamide Gels
2.5.5 Western Blot Analysis
2.6 Mass-Spectrometric Analysis of Antibodies Produced in Regenerated Plants
2.6.1 Recombinant Antibody Expression
2.6.2 Antibody Purification
2.6.3 SDS-PAGE
2.6.4 LC-ESI-MS
2.7 Mass-Spectrometric Analysis of Endogenous Leaf Proteins of Regenerated Plants
2.7.1 MALDI-TOF-MS
2.8 Manual Crossing of Plants
3 Methods
3.1 Generation of Transgenic Plants
3.1.1 Target Gene Resequencing
Primer Design
Spin-Column Extraction of Plant Genomic DNA
Polymerase Chain Reaction of Genomic DNA
Agarose Gel Electrophoresis
Spin-Column Purification of Linear DNA Fragments
Sanger Sequencing
Analysis of Sanger Sequencing Results
3.1.2 gRNA Cloning and Testing
gRNA Design
Cloning of gRNA Test Constructs
Transformation of Chemically Competent E. coli
Colony PCR of Transformed E. coli
Liquid Cultivation of E. coli
Plasmid Preparation from E. coli
Preparation of Competent A. tumefaciens Cells for Electroporation
Transformation of Electrocompetent A. tumefaciens
Colony PCR of Electroporated A. tumefaciens
Liquid Cultivation of A. tumefaciens
Agroinfiltration
Analysis of gRNA Efficiency
3.1.3 Stable Transformation of Plants
Cloning of the Transformation Construct
Agrotransformation and Agroinfiltration
Plant Transformation and Regeneration
3.2 Screening of Transgenic Plants
3.2.1 Genetic Analysis of Regenerated Plants
Fast and Easy Extraction of Plant Genomic DNA
Target Gene Amplification for Direct Sequencing
Clean-Up of PCR Products
Sanger Sequencing of Purified PCR Products
3.2.2 Immunoblot Analysis of Regenerated Plants
Dot Blot Analysis
Protein Extraction for SDS-PAGE
SDS-PAGE
Microwave-Assisted Coomassie Staining of Acrylamide Gels
Western Blot Analysis
3.2.3 Mass-Spectrometric Analysis of Antibodies Produced in Genome-Edited Plants
Recombinant Antibody Expression
Antibody Purification
SDS-PAGE
LC-ESI-MS
3.2.4 Mass-Spectrometric Analysis of Endogenous Leaf Proteins of Regenerated Plants
MALDI-TOF-MS
3.3 Crossing
3.3.1 Manual Crossing of N. benthamiana Plants
4 Notes
References
Chapter 15: A Bioluminescent Agrobacterium tumefaciens for Imaging Bacterial Metabolic Activity in Planta
1 Introduction
2 Materials
2.1 Plants
2.2 AgroLux
2.3 Laboratory Tools and Materials
2.4 Buffers and Other Solutions
2.5 Software
3 Methods
3.1 AgroLux Competent Cells and Transformation
3.1.1 AgroLux Competent Cells
3.1.2 AgroLux Transformation
3.2 Agroinfiltration
3.2.1 Bacterial Growth and Leaf Agroinfiltration
3.2.2 Post-Infiltration Incubation Conditions
3.3 AgroLux Luminescence Imaging and Data Analysis
3.3.1 Whole Leaf Imaging
3.3.2 Image Intensity Processing Using ImageJ
3.3.3 Apply a Look-Up Table (LUT) to Color Images Using ImageJ
3.3.4 Leaf Discs Data Acquisition
4 Notes
References
Chapter 16: Statistical Designs to Improve Downstream Processing
Abbreviations
1 Introduction
2 Materials
3 Methods
3.1 Plan a Design of Experiments Strategy
3.1.1 Define the Context, Goal, and General Procedure of the Experiment
3.1.2 Identify Relevant Factors and Responses for Inclusion in the Design
3.2 Prepare a Specific Experimental Design
3.2.1 Set Up a Factorial Screening Design
3.2.2 Set Up a Response Surface Design
3.3 Conduct the Experiment
3.4 Analyze the Data
3.4.1 Analyze Factorial Screening Designs
3.4.2 Analyze Response Surface Designs
4 Notes
References
Part IV: Regulatory Issues
Chapter 17: Regulation of Molecular Farming Products
1 Introduction to Molecular Farming
2 Approval of Pharmaceutical Products
2.1 General Regulations for the Approval of Pharmaceutical Products
2.2 Accelerated Approval Routes for Pharmaceuticals
2.3 Special Regulations Applicable to Molecular Farming Products
2.4 Molecular Farming: Biological Raw Materials
2.5 Molecular Farming: Upstream Production
2.6 Molecular Farming: Downstream Processing
3 Other Regulated Products
3.1 Medical Devices
3.2 Cosmetics
3.3 Veterinary Products
3.4 Research Reagents
4 Environmental Aspects of Molecular Farming
5 Conclusions
6 Notes
References
Chapter 18: Freedom to Operate Analysis of Molecular Farming Projects
1 Introduction
2 Freedom-to-Operate Analysis
3 Process and Methodology of FTO Analysis
4 Illustrative Example of an FTO Analysis
References
Index