Recombinant Glycoproteins: Methods and Protocols (Methods in Molecular Biology, 2762)

Preface
Contents
Contributors
Part I: Viral Surface Glycoproteins
	Chapter 1: Production and Purification of Hantavirus Glycoproteins in Drosophila melanogaster S2 Cells
		1 Introduction
			1.1 Hantavirus Glycoproteins
			1.2 Production of Hantavirus Glycoproteins
		2 Materials
			2.1 Plasmids
			2.2 Cells and Medium
			2.3 Other Materials
			2.4 Protein Purification Buffers
		3 Methods
			3.1 Thawing S2 Cells
			3.2 Passaging of S2 Cells
			3.3 Transfection and Selection of Stable Cell Lines
			3.4 Expansion of Cell Culture
			3.5 Protein Purification
			3.6 Freezing S2 Cells
		4 Notes
		References
	Chapter 2: Production and Purification of Filovirus Glycoproteins
		1 Introduction
		2 Materials
			2.1 S2 Cell Culture
			2.2 Transfection and Harvest
			2.3 Purification with Strep-Tactin Resin
			2.4 SDS-Page
		3 Methods
			3.1 S2 Cell Culture
			3.2 Transient Transfection (Step 1)
			3.3 Stable Transfection and Selection (Step 2)
			3.4 Harvest (Step 3)
			3.5 Purification with Strep-Tactin Resin (Step 4)
			3.6 SDS-Page
		4 Notes
		References
	Chapter 3: Modification of N-Linked Glycan Sites in Viral Glycoproteins
		1 Introduction
		2 Materials
			2.1 Reagents
			2.2 Buffers
			2.3 Cell Lines and Media
			2.4 Equipment
		3 Methods
			3.1 In Silico Protein Analysis
			3.2 Design of Mutagenesis Primers
			3.3 Site-Directed Mutagenesis
				3.3.1 Method 1: Quick Change Protocol
				3.3.2 Method 2: Multi-fragment Assembly Mutagenesis
			3.4 Assessment of NGS Present on Parental and Mutant Constructs and Enzymatic Removal of Glycans
			3.5 Production of VSV Pseudovirions Bearing Wild-type or Mutant EBOV GP and the Effect of Glycans on Viral Glycoprotein-mediat...
		4 Notes
		References
	Chapter 4: Production of Influenza Virus Glycoproteins Using Insect Cells
		1 Introduction
		2 Materials
			2.1 General Supplies (Used Throughout Protocol)
			2.2 Polymerase Chain Reaction and Colony  PCR
			2.3 PCR Purification Kit, Gel Extraction Kit, and Spin Miniprep  Kits
			2.4 Restriction Digestion, Ligation, and Transformation
			2.5 Midiprep  Kit
			2.6 Transfection
			2.7 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS PAGE)
			2.8 Western Blotting
			2.9 Protein Purification
		3 Methods
			3.1 Template/Primer Ordering and  PCR
			3.2 PCR Purification (Using QIAquick PCR Purification Kit)
			3.3 Restriction Digestion of Vector and Gene of Interest (Qiagen)
			3.4 Gel Extraction (Using Qiagen Gel Extraction Kit)
			3.5 Ligation, Transformation into XL10-Gold Competent Cells (Agilent), and Plating
			3.6 Colony PCR, and Miniprep Set  Up
			3.7 Miniprep (Using Qiagen Kit)
			3.8 Transformation into DH10Bac (Invitrogen) Competent Cells
			3.9 Midiprep (Using Invitrogen Kit)
			3.10 Sf9 Cell Transfection
			3.11 Western Blotting
				3.11.1 Separation
				3.11.2 Transfer
				3.11.3 Detection
			3.12 Sf9 Infection and P0 Amplification
			3.13 Cell Harvesting and Baculovirus Infection
			3.14 Nickel Bead Incubation
			3.15 Filtration, Washing, Elution, and Concentration
			3.16 Protein Harvesting, Quantification, and Aliquoting
			3.17 SDS PAGE for Quality Check
		4 Notes
		References
	Chapter 5: SARS-CoV-2S-Protein-Ace2 Binding Analysis Using Surface Plasmon Resonance
		1 Introduction
		2 Materials
		3 Methods
			3.1 Creation of the SPR Capture Antibody Surface
			3.2 Generating the Ace2 Binding Sensorgrams
			3.3 Data Analysis
		4 Notes
		References
	Chapter 6: A Biosensor Assay Based on Coiled-Coil-Mediated Human ACE2 Receptor Capture for the Analysis of Its Interactions wi...
		1 Introduction
		2 Materials
			2.1 Cell Thawing
			2.2 Plasmid DNA Preparation (Maxiprep)
			2.3 Polyethylenimine MAX Solution
			2.4 BalanCD CHO Feed 4
			2.5 Transfection of CHO Cells with PEI MAX
			2.6 Purification of His-Tagged Proteins
			2.7 Purification of Strep-Tagged Proteins
			2.8 Purification of FLAG-Tagged Proteins
			2.9 SEC Purification
			2.10 Functionalization of SPR Biosensor Chip
			2.11 SPR Assay for the ACE2-RBD Interaction Monitoring
		3 Methods
			3.1 Cell Thawing and Maintenance
			3.2 Plasmid DNA Preparation
			3.3 Linear PEI-MAX (1 mg/mL Solution)
			3.4 BalanCD CHO Feed 4 Preparation (0.8x Concentration)
			3.5 Transfection of CHO Cells
			3.6 Purification of Secreted His-Tagged Proteins
			3.7 Purification of Secreted Strep-Tagged Proteins
			3.8 Purification of FLAG-Tagged Proteins
			3.9 SEC Purification
			3.10 Functionalization of SPR Biosensor Chip
			3.11 SPR Assay for ACE2-RBD Interaction Analysis
			3.12 SPR Data Analysis
		4 Notes
		References
Part II: Protozoal and Nematode Glycoproteins and Polysaccharide Conjugates
	Chapter 7: Production and Purification of Plasmodium Circumsporozoite Protein in Lactococcus lactis
		1 Introduction
		2 Materials
			2.1 General Supplies
			2.2 Subcloning of Expression Vector and Transformation
				2.2.1 Prepared Solutions
				2.2.2 Other Reagents and Materials
			2.3 Small-Scale Expression and Cell Bank
			2.4 Fermentation
			2.5 Protein Purification
		3 Methods
			3.1 Subcloning of Expression Vector for the Recombinant Protein and Transformation into L. Lactis
				3.1.1 Subcloning
				3.1.2 Transformation
			3.2 Small-Scale Culture for Protein Production and Cell Banking
			3.3 Large-Scale Cell Culture (Fermentation) for Protein Production
			3.4 Protein Purification: Affinity Chromatography
		4 Notes
		References
	Chapter 8: Analysis of Caenorhabditis Protein Glycosylation
		Abbreviations
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 Reagents, Buffers, and Columns (See Notes 1 and 2)
				2.2.1 Disruption of Biological Material and SDS-PAGE Sample Preparation
				2.2.2 SDS-PAGE and Western Blotting
				2.2.3 N-glycome Release and Analysis
				2.2.4 O-glycome Release and Analysis
				2.2.5 Glycosaminoglycan Release and Analysis
				2.2.6 Glycan Data Analysis
		3 Methods
			3.1 Sample Preparation and Glycoepitope Recognition
				3.1.1 Sample Preparation for Glycoprotein Analysis (See Notes 1 and 2)
				3.1.2 SDS-PAGE and Western Blotting
			3.2 Glycan Release and Analysis
				3.2.1 N-glycan Release, Fractionation, and Analysis (See Notes 5-8)
				3.2.2 O-glycan Release (See Note 9)
				3.2.3 Glycosaminoglycan Release (See Note 10)
		4 Notes
		References
	Chapter 9: Use of Reductive Amination to Produce Capsular Polysaccharide-Based Glycoconjugates
		1 Introduction
		2 Materials
			2.1 Antigens
			2.2 Activation of CPS
			2.3 Preparation of CRM197
			2.4 Conjugation Reaction
		3 Methods
			3.1 Activation of CPS
			3.2 Preparation of CRM197
			3.3 Conjugation of CPS to CRM197
		4 Notes
		References
Part III: Mammalian Glycoproteins
	Chapter 10: Overexpression and Purification of Mitogenic and Metabolic Fibroblast Growth Factors
		1 Introduction
		2 Materials
			2.1 Buffers
			2.2 Media and Cells
			2.3 Solutions
			2.4 Equipment
		3 Methods
			3.1 Construction of an FGF1 Gene Containing Expression Vector and Transformation of BL21 Star E. coli Competent Cells with the...
			3.2 Transforming Expression Vectors Contain Metabolic FGFs Genes into BL21 Star E. coli Competent Cells with the Constructed V...
			3.3 Preparation of Starter Culture
			3.4 Preparation of Small-Scale Bacterial Culture for Protein Overexpression
			3.5 Preparation of Large-Scale Bacterial Culture for Protein Overexpression
			3.6 FGF1 Purification Using Heparin Affinity Chromatography
				3.6.1 Column Packing
				3.6.2 Bacterial Cell Lysis and Loading
				3.6.3 Using Heparin-Affinity Chromatography to Purify FGF1
			3.7 Immobilized Metal Affinity Chromatography is Used to Purify Metabolic FGFs
				3.7.1 Column Packing
				3.7.2 Bacterial Cell Lysis and Loading
				3.7.3 Purification of Metabolic FGFs Using IMAC
			3.8 Denaturing (SDS) Polyacrylamide Gel Electrophoresis (PAGE)
				3.8.1 Gel Casting
				3.8.2 Stacking  Gel
				3.8.3 Sample Preparation and Loading
				3.8.4 Gel Running
			3.9 Transferring the Protein from the Gel to the Membrane/Western  Blot
				3.9.1 Set Up Blotting Sandwich and Transfer
			3.10 Isothermal Titration Calorimetry (ITC)
			3.11 Differential Scanning Calorimetry
				3.11.1 DSC  Run
			3.12 Circular Dichroism
				3.12.1 Recording a CD Spectrum
			3.13 Intrinsic Fluorescence Spectroscopy
				3.13.1 Preparation
				3.13.2 Settings
				3.13.3 Measurements
				3.13.4 Analysis
			3.14 Standard Urea Denaturation Assay
			3.15 Alternative Urea Denaturation Assay/Sypro Orange Assay
			3.16 Cell Proliferation Assay
				3.16.1 Complete Media (CM) Preparation
				3.16.2 Expanding Cells
				3.16.3 Detaching Cells
				3.16.4 Cell Proliferation Assay
		4 Notes
		References
	Chapter 11: Production and Purification of Antibodies in Chinese Hamster Ovary Cells
		1 Introduction
		2 Materials
			2.1 Cell Maintenance
			2.2 Transient Transfection
			2.3 Harvesting Cells
			2.4 Antibody Purification
		3 Methods
			3.1 Cell Maintenance
			3.2 Transient Transfection
			3.3 Harvesting Cells
			3.4 Antibody Purification
		4 Notes
		References
	Chapter 12: Mammalian Antigen Display for Pandemic Countermeasures
		1 Introduction
		2 Materials
			2.1 Media, Strains, Plasmids
			2.2 Generating Antigen Libraries
				2.2.1 Golden Gate Assemblies
				2.2.2 Saturation Mutagenesis Library Generation
			2.3 Expression of Surface-Displayed Antigens
				2.3.1 Transient Transfection of HEK293Ts
			2.4 Immunofluorescence Microscopy
			2.5 Cleavage and Purification of Surface-Expressed Antigens
			2.6 Flow Cytometry
			2.7 Fluorescence-Assisted Cell Sorting
			2.8 Data Analysis
		3 Methods
			3.1 Generating Antigen Libraries
				3.1.1 Golden Gate Assemblies
				3.1.2 Saturation Mutagenesis Libraries
			3.2 Antigen Expression
				3.2.1 Transient Transfection of HEK293Ts
			3.3 Immunofluorescence Microscopy
				3.3.1 Cell Preparation for Immunostaining
				3.3.2 Multi-color Immunostaining and Imaging
			3.4 Cleavage and Purification of Surface-expressed Antigens
			3.5 Flow Cytometry
				3.5.1 Collect Cells
				3.5.2 Immunostaining
			3.6 Flow Cytometry
				3.6.1 Flow Cytometry Analysis
				3.6.2 Fluorescence-Assisted Cell Sorting (FACS)
				3.6.3 Collect Cells
				3.6.4 Immunostaining
				3.6.5 Cell Sorting
				3.6.6 Data Analysis
		4 Notes
		References
Part IV: Analysis of Glycoproteins
	Chapter 13: Analysis of Native and Permethylated N-Glycan Isomers Using MGC-LC-MS Techniques
		1 Introduction
		2 Materials
			2.1 Biological Reagents
			2.2 MGC Column Materials
			2.3 Other Materials
			2.4 Instruments
		3 Methods (See Note 1)
			3.1 MGC Column Preparation
			3.2 PNGase F Digestion
			3.3 SPE-C18 Cleanup
			3.4 Reduction
			3.5 Permethylation (See Note 6)
			3.6 nanoLC Conditions (Native)
			3.7 nanoLC Conditions (Permethylated)
			3.8 MS Conditions (Native)
			3.9 MS Conditions (Permethylated)
			3.10 Data Analysis
		4 Notes
		References
	Chapter 14: Targeted Glycoproteomics Analysis Using MRM/PRM Approaches
		1 Introduction
		2 Materials
			2.1 Model Glycoproteins
			2.2 Reagents
		3 Methods
			3.1 Enzymatic Digestion of N-glycopeptides
			3.2 Enzymatic Digestion of O-glycopeptides
			3.3 LC Instrumentation
			3.4 MS System and MRM Parameters
			3.5 MS System and PRM Parameters
			3.6 Full MS for MRM/PRM Analysis
			3.7 MRM/PRM Acquisition Parameters
			3.8 MRM Transitions
			3.9 PRM Transitions
			3.10 MRM/PRM Quantitation
		4 Notes
		References
	Chapter 15: Targeted Analysis of Permethylated N-Glycans Using MRM/PRM Approaches
		1 Introduction
		2 Materials
			2.1 Model Glycoproteins
			2.2 Cell Lines
			2.3 Reagents
		3 Methods
			3.1 N-Glycan Release, Purification, Reduction, and Permethylation
			3.2 LC Conditions
			3.3 MS-MRM Conditions
			3.4 MS-PRM Conditions
			3.5 Full MS for MRM/PRM Analysis
			3.6 MRM/PRM Acquisition Parameters for Transition List
			3.7 MRM Transitions
			3.8 PRM Transitions
			3.9 MRM/PRM Quantitation
		4 Notes
		References
	Chapter 16: Hydrophilic Interaction Liquid Chromatography (HILIC) Enrichment of Glycopeptides Using PolyHYDROXYETHYL A
		1 Introduction
		2 Materials
			2.1 Cells Protein Extraction
			2.2 Tissue Protein Extraction
			2.3 Depletion of Abundant Proteins from Blood Serum
			2.4 Buffer Exchange
			2.5 Protein Assay
			2.6 Tryptic Digestion
			2.7 C18 Desalting
			2.8 Glycopeptides Enrichment Using PolyHYDROXYETHYL A (HILIC)
			2.9 LC-MS/MS Analysis
		3 Methods
			3.1 Protein Extraction
			3.2 Cells Protein Extraction
			3.3 Tissue Protein Extraction
			3.4 Blood Serum Protein Extraction
			3.5 Depletion of Abundant Proteins
			3.6 Buffer Exchange
			3.7 Tryptic Digestion
			3.8 C18 Desalting
			3.9 Glycopeptides Enrichment Using PolyHYDROXYETHYL A (HILIC)
			3.10 LC-MS/MS Analysis
			3.11 LC Condition
			3.12 MS Condition
			3.13 Data Processing
		4 Notes
		References
	Chapter 17: O-Glycoproteomics Sample Preparation and Analysis Using NanoHPLC and Tandem MS
		1 Introduction
		2 Materials
			2.1 Solutions for Enzymatic Digestion
			2.2 Mobile Phase for RPLC Separation
		3 Methods
			3.1 Tryptic Digestion
			3.2 O-Glycoprotease Digestion
			3.3 nanoLC Conditions
			3.4 MS Conditions
			3.5 Data Processing
		4 Notes
		References
Part V: Considerations and Alternatives for Glycoprotein Production and Purification
	Chapter 18: Solubilization of Oligomeric Cell-Free Synthesized Proteins Using SMA Copolymers
		1 Introduction
		2 Materials
			2.1 Cell-Free Synthesis Based on Sf21 Lysate
				2.1.1 Quantitative Protein Analysis
				2.1.2 Qualitative Protein Analysis
			2.2 Solubilization of Cell-Free Expressed Membrane Proteins
			2.3 Particle Size Measurement
			2.4 Complementation Assay
		3 Methods
			3.1 Cell-Free Protein Synthesis
				3.1.1 Quantitative Analysis
				3.1.2 Qualitative Analysis and Deglycosylation Assay
			3.2 Solubilization of Vesicular Fraction Using SMA Copolymers
				3.2.1 Ultrafiltration
				3.2.2 Particle Size Measurement of SMA Lipid Particles
			3.3 Complementation Assay for Fluorescence Detection of Protein-Protein Interactions
		4 Notes
		References
	Chapter 19: Cell-Free Systems for the Production of Glycoproteins
		1 Introduction
		2 Materials
			2.1 Bacterial Strains and Plasmids
			2.2 Buffers, Media, and Chemicals
			2.3 CFPS Reagents
			2.4 Equipment and Atypical Lab Materials
		3 Methods
			3.1 S12 Lysate Preparation (Fig. 2a)
				3.1.1 Media Preparation and Inoculation of Overnight Culture (see Note 1)
				3.1.2 Lysate Strain Growth and Harvest
				3.1.3 Resuspension, Lysis, and Centrifugation
				3.1.4 CFPS Reaction and Optimization of Magnesium in Lysate
			3.2 Solvent Extraction of LLOs from E. coli (Fig. 3a)
				3.2.1 Media Preparation and Inoculation of Overnight (Day 1)
				3.2.2 Cell Growth and Harvest (Days 2-3)
				3.2.3 LLO Extraction (Day 4)
				3.2.4 LLO Recovery (Day 5)
			3.3 Expression, Membrane Solubilization, and Purification of CjPglB (Fig. 4a)
				3.3.1 Media Preparation and Inoculation of Overnight Culture (Day 1)
				3.3.2 Cell Growth and Protein Induction (Days 2-3)
				3.3.3 Isolation and Extraction of Membrane Protein (Day 3)
				3.3.4 Protein Purification Using Affinity Column (Day 4)
			3.4 IVG Utilizing CFPS Reaction Sample
		4 Notes
		References
	Chapter 20: Considerations for Glycoprotein Production
		1 Introduction
		2 Expression Systems
			2.1 Bacterial Expression Systems
			2.2 Yeast Expression Systems
			2.3 Insect Cells and Baculovirus Systems
			2.4 Cell-Free Systems
			2.5 Mammalian Cell Systems
				2.5.1 Chinese Hamster Ovary Cells
				2.5.2 Human Endothelial Kidney (HEK) 293 Cells
				2.5.3 Murine Hybridoma Cell Lines
		3 Additional Considerations
			3.1 Culture Conditions in Mammalian Cell Culture
			3.2 O-Linked Glycosylation
			3.3 Purification of Glycoproteins
			3.4 Protein Sequence Modifications
		4 Conclusions
		References
Index