Lectin Purification and Analysis: Methods and Protocols (Methods in Molecular Biology, 2132)

Preface
Contents
Contributors
Chapter 1: Structural Database for Lectins and the UniLectin Web Platform
	1 Introduction
	2 Existing Lectin Databases
	3 UniLectin3D, Database of Curated Lectin 3D Structure, and Their Interacting Ligands
		3.1 Searching by Keywords
		3.2 Searching by Kingdom Order, Carbohydrate-Binding Site Class, and Species Family
		3.3 Searching by Monosaccharide and Associate IUPAC Sequence
		3.4 Searching by Fold
		3.5 Advanced Search: Searching by Multiple Criteria
		3.6 Lectin Sequence Detailed Interface
		3.7 X-Ray Structure Detailed Interface
	4 PropLec: Database of Structure-Based Predicted β-Propellers
		4.1 Searching by Keywords and by Family
		4.2 Searching by Number of Blades in the Propeller
		4.3 Searching by Phylum
		4.4 Advanced Search
		4.5 Detailed Results
		4.6 Searching by Other Pfam Functional Domains
	5 Conclusion and Discussion
	References
Chapter 2: Purification of Sugar-Binding Peptides from L-Type Lectins
	1 Introduction
	2 Materials
		2.1 Preparation of Sugar-Immobilized Sepharose
		2.2 Purification of L-Type Lectins
		2.3 Purification and Identification of the Sugar-Binding Peptide of L-Type Lectins
	3 Methods
		3.1 Preparation of Sugar-Immobilized Sepharose
		3.2 Purification of L-Type Lectins
		3.3 Purification and Identification of the Sugar-Binding Peptides of L-Type Lectins
	4 Notes
	References
Chapter 3: Recombinant Expression and Purification of Animal Intracellular L-Type Lectins
	1 Introduction
	2 Materials
		2.1 Construction of Expression Vectors
		2.2 Expression and Purification of Animal L-Type Lectins
	3 Methods
		3.1 Soluble Expression and Purification of Human VIP36-CRD
		3.2 Refolding and Purification of ERGIC-53-CRD
		3.3 Refolding and Purification of VIPL-CRD
	4 Notes
	References
Chapter 4: Frontal Affinity Chromatography: A Highly Suitable Retardation Phenomenon-Based Research Tool for Analyzing Weak In...
	1 Introduction
	2 Peculiar Characteristics of Lectin-Glycan Interactions
	3 Significance of Fuzzy and Weak Interactions in Biological Systems
	4 FAC: A Powerful Analytical Tool for Weak Interactions
	5 FAC Viewed as Retardation Analysis of Bioaffinity
	6 Two Diverse Aspects of FAC
	7 Concluding Remarks
	References
Chapter 5: Metazoan Soluble β-Galactoside-Binding Lectins, Galectins: Methods for Purification, Characterization of Their Carb...
	1 Introduction
	2 Materials
		2.1 Purification of Recombinant Galectins
		2.2 Carboxymethylation of Galectin-1
		2.3 Hemagglutinin Assay
		2.4 Biotinylation Using Primary Amine Residues of Galectin
		2.5 Biotinylation Using Cysteine Residues of Galectin-1
		2.6 Enzyme-Linked Lectin Sorbent Assay
		2.7 Surface Plasmon Resonance (SPR) Assay
		2.8 Staining with Galectin
		2.9 Immunostaining of Galectins
		2.10 Special Equipment
	3 Methods
		3.1 Purification of a Galectin
		3.2 Carboxymethylation of Galectin-1
		3.3 Quality Control of Purified Galectin by Hemagglutination Assay
			3.3.1 Preparation of Red Blood Cells (RBC)
			3.3.2 Calibration of RBC
			3.3.3 Hemagglutination Assay
		3.4 Biotinylation of Galectins Using Primary Amine Residues of a Galectin
		3.5 Biotinylation of Galectin-1 Using Cysteine Residues
		3.6 Carbohydrate-Binding Specificity Analysis of Galectin: Enzyme-Linked Lectin Sorbent Assay
		3.7 Carbohydrate-Binding Specificity Analysis of Galectin: SPR Assay
		3.8 Visualization of Ligands of a Galectin Expressed in Cells (Lectin Staining)
		3.9 Detection of a Galectin Expressed in Cells Using an Antibody against Galectin
	4 Notes
	References
Chapter 6: Expression, S-Nitrosylation, and Measurement of S-Nitrosylation Ratio of Recombinant Galectin-2
	1 Introduction
	2 Materials
		2.1 Preparation of Recombinant Gal-2
		2.2 S-Nitrosylation of Recombinant Gal-2
		2.3 Measuring S-Nitrosylation by Saville-Griess Assay
	3 Methods
		3.1 Expression of Recombinant Gal-2 in Escherichia coli
		3.2 Purification of Recombinant Gal-2 by Affinity Chromatography
		3.3 S-Nitrosylation of Recombinant Gal-2
		3.4 Measuring S-Nitrosylation of Gal-2 by Saville-Griess Assay
	4 Notes
	References
Chapter 7: Expression and Purification of Full-Length and Domain-Fragment Recombinant Pentraxin 3 (PTX3) Proteins from Mammali...
	1 Introduction
	2 Materials
		2.1 Plasmids for Recombinant PTX3 Protein Expression
		2.2 Cell Culture for the Mammalian Expression System
		2.3 Expression of the N-Terminal Domain of PTX3 by Bacterial Cells
		2.4 Purification of Recombinant PTX3 Proteins from Mammalian Cells with a 6xHis Tag Affinity Column
		2.5 Purification of Recombinant N-Terminal Domain PTX3 Proteins from Bacterial Cells with a 6xHis Tag Affinity Column
	3 Methods
		3.1 Establishment of Recombinant PTX3 Expressing Stable Mammalian Cell Lines
		3.2 Large-Scale Culture of PTX3-Expressing Stable Cell Lines
		3.3 Expression of the N-Terminal Domain of PTX3 by Bacterial Cells
		3.4 Purification of Recombinant PTX3 Proteins from the Mammalian Cell Culture Supernatant with a 6xHis Tag Affinity Column
		3.5 Purification of Recombinant N-Terminal Domain PTX3 Proteins from Bacterial Cells with a 6xHis Tag Affinity Column
	4 Notes
	References
Chapter 8: Identification of Siglec Cis-Ligands by Proximity Labeling
	1 Introduction
	2 Materials
		2.1 Proximity Labeling Using Tyramide
		2.2 Flowcytometry Analysis
		2.3 Immunoprecipitation, SDS-PAGE, and Western Blotting
	3 Methods
		3.1 Proximity Labeling
		3.2 Flow Cytometry Analysis of Labeling Efficacy of Cell Surface Molecules
		3.3 SDS-PAGE and Western Blotting for Biotinylated Proteins
		3.4 Immunoprecipitation of Biotinylated Proteins, SDS-PAGE, and Western blotting to Identify Cis-Ligands
	4 Notes
	References
Chapter 9: Preparation of Recombinant Siglecs and Identification of Their Ligands
	1 Introduction
	2 Materials
		2.1 Preparation of Recombinant Siglec-Fc
			2.1.1 Equipment
			2.1.2 Reagents and Consumables
		2.2 Flow Cytometry Using Siglec-Fc as a Probe
			2.2.1 Equipment
			2.2.2 Reagents and Consumables
		2.3 Proximity Labeling Using Siglec-Fc as a Probe
			2.3.1 Equipment
			2.3.2 Reagents and Consumables
	3 Methods
		3.1 Preparation of Recombinant Siglec-Fc
			3.1.1 Maintenance of Expi293F Cells
			3.1.2 Transfection
			3.1.3 Purification
		3.2 Flow Cytometry Using Siglec-Fc as a Probe
		3.3 Proximity Labeling with Siglec-Fc
			3.3.1 Biotin-Tyramide Labeling of Ligand Proteins
			3.3.2 Affinity Purification of Biotinylated Proteins
			3.3.3 Evaluation of Biotin Labeling
	4 Notes
	References
Chapter 10: Purification, Quantification, and Functional Analysis of Collectins
	1 Introduction
	2 Materials
		2.1 Buffers and Assay Solutions
	3 Methods
		3.1 Purification of Native and Recombinant Human MBL
			3.1.1 In the Case of Native Human MBL
			3.1.2 In the Case of Recombinant Human MBL from a Mammalian Expression System
			3.1.3 Purification Step
		3.2 Preparation of the CL-K1-Rich Fraction from Human Serum or Plasma
		3.3 Quantification of Human MBL by Sandwich ELISA
		3.4 Saccharide-Binding Assay
			3.4.1 To Assay Sugar Selectivity
		3.5 Assays of Biological Function
			3.5.1 Complement-Dependent Passive Hemolysis
			3.5.2 Hemagglutination Inhibition (HI) Test
			3.5.3 Neutralization (NT) Test
			3.5.4 The Immunoplaque Assay for Viral Growth Inhibition
	4 Notes
	References
Chapter 11: Selectin-Binding Assay by Flow Cytometry
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Flow Cytometry
	3 Methods
		3.1 Cell Preparation
		3.2 Flow Cytometry
		3.3 Neutralization of sLex/a Using Monoclonal Antibodies
	4 Notes
	References
Chapter 12: Direct Binding Analysis Between C-Type Lectins and Glycans Using Immunoglobulin Receptor Fusion Proteins
	1 Introduction
	2 Materials
		2.1 Transfection
		2.2 Evaluation of Concentration
		2.3 Molecular Weight Analysis by Western Blotting
		2.4 Molecular Weight Analysis Using Coomassie Brilliant Blue
		2.5 Binding Assay
		2.6 Staining
	3 Methods
		3.1 Transfection
		3.2 Evaluation of Concentration
		3.3 Molecular Weight Analysis by Western Blotting
		3.4 Molecular Weight Analysis Using Coomassie Brilliant Blue
		3.5 Binding Assay
		3.6 Staining
	4 Notes
	References
Chapter 13: Expression and Characterization of Hyaluronan-Binding Protein Involved in Hyaluronan Depolymerization: HYBID, Alia...
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Transfection of Expression Vector and Positive Clones Selection
		2.3 HA Labeling
		2.4 Binding Assay
	3 Methods
		3.1 Preparation of HYBID Stable Transfectants (HYBID/HEK293 Cells)
		3.2 Preparation of [3H]-Labeled High-Molecular-Weight HA ([3H]HA)
		3.3 Depolymerization of [3H]-Labeled HA ([3H]HA) and FA-HA H1
			3.3.1 Depolymerization of [3H]HA
			3.3.2 Depolymerization of FA-HA H1
		3.4 Binding Assay for HYBID to Glycosaminoglycans (GAGs) by Coprecipitation with CPC
	4 Notes
	References
Chapter 14: Paracoccin: Purification and Validation of Its Lectin and Enzymatic Properties
	1 Introduction
	2 Materials
		2.1 Crude Extract of P. brasiliensis Yeasts
		2.2 Protein Expression by E. coli
		2.3 Protein Expression by P. pastoris
		2.4 PCN Purification by Affinity to GlcNAc
		2.5 PCN Purification by Affinity to Particulate Chitin
		2.6 Assay for PCN Binding to Laminin
		2.7 N-Acetylglucosaminidase (NAGase) Activity Assay
	3 Methods
		3.1 Preparation of the Crude Extract of P. brasiliensis
		3.2 PCN Expression in E. coli and Purification Process
		3.3 PCN Expression in P. pastoris and Purification Guidelines
		3.4 Purification of PCN by Affinity to Immobilized GlcNAc
		3.5 Purification of PCN by Affinity to Particulate Chitin
		3.6 Lectin Activity: Assay for PCN Binding to Laminin
		3.7 Enzymatic Properties: NAGase Activity Assay
	4 Notes
	References
Chapter 15: In Vitro Mannosidase Assay of EDEMs: ER Degradation-Enhancing α-Mannosidase-Like Proteins
	1 Introduction
	2 Materials
		2.1 Preparation of Buffers and Reagents
		2.2 Preparation of Plasmids and Poly-l-Lysine-Coated Dishes
	3 Methods
		3.1 Cell Culture and Transfection
		3.2 Preparation of Recombinant Proteins from Cultured Cells
		3.3 In Vitro Mannosidase Assay
	4 Notes
	References
Chapter 16: Galactose-Specific, Hemolytic Lectin CEL-III from Cucumaria echinata
	1 Introduction
	2 Materials
		2.1 Reagents/Solutions/Materials
		2.2 Preparation of the Carbohydrate-Immobilized Microplate
		2.3 Preparation of the Colloidal Gold Solution
	3 Methods
		3.1 Purification of CEL-III
		3.2 Microplate Assay for the Binding of CEL-III to Lactose
	4 Notes
	References
Chapter 17: Simple and Rapid Detection of Glycoforms by ``Lectin Inhibition´´ Assay
	1 Introduction
	2 Materials
		2.1 Periodate Treatment of Antibody
		2.2 Preparation of Tf Glycoforms
		2.3 SSA Inhibition in ELISA
	3 Methods
		3.1 Periodate Treatment of Antibody
		3.2 Preparation of Tf Glycoforms
		3.3 SSA Inhibition in ELISA
	4 Notes
	References
Chapter 18: Glycoform-Specific Visualization in Immunohistochemistry by ``Lectin Inhibition´´
	1 Introduction
	2 Materials
		2.1 Periodate Treatment of Antibody
		2.2 SSA Inhibition with Tissue Sections
		2.3 SSA Staining with Sialidase-Treated Sections
		2.4 SSA Inhibition with Sialidase-Treated Sections
	3 Methods
		3.1 Periodate Treatment of Antibody
		3.2 SSA Inhibition with Tissue Sections
		3.3 SSA Staining with Sialidase-Treated Sections
		3.4 SSA Inhibition with Sialidase-Treated Sections
	4 Notes
	References
Chapter 19: Botulinum Hemagglutinin: Critical Protein for Adhesion and Absorption of Neurotoxin Complex in Host Intestine
	1 Introduction
	2 Structure of Botulinum HA
	3 Carbohydrate Binding of HA1 and HA3
	4 Differences in Lectin Activities Between Serotypes
	5 L-PTC/A Is Absorbed from M-Cells in the Mouse Intestine by the Carbohydrate-Binding Activity of HA
	6 Apical Cell Surface Binding of HA of L-PTC/A-D
	7 Basolateral Cell Surface Binding of HA/[A, B]
	8 Multivalency Effect of HA
	9 Conclusion
	References
Chapter 20: Functional Analysis of Botulinum Hemagglutinin (HA)
	1 Introduction
	2 Materials
		2.1 Generation of Recombinant HA
		2.2 Cell ELISA
		2.3 HA Pull-Down Assay
		2.4 In Situ Loop Assay
		2.5 Immunofluorescence Staining
	3 Methods
		3.1 Generation of Recombinant HA
		3.2 Cell ELISA: Binding Assay Using Epithelial Cells
		3.3 HA Pull-Down Assay: Binding Assay to Human Fc-Tagged Glycoprotein
		3.4 Localization of HA in the Mouse Intestine
			3.4.1 Mouse In Situ Loop Assay
			3.4.2 Immunofluorescence Staining
	4 Notes
	References
Chapter 21: Purification and Functional Characterization of the Effects on Cell Signaling of Mytilectin: A Novel β-Trefoil Lec...
	1 Introduction
	2 Materials
		2.1 Mussels
		2.2 Lectin Purification
		2.3 Cell Culture and Cell Growth Assay
		2.4 Antibodies and Western Blotting
	3 Methods
		3.1 Purification of MytiLec-1
		3.2 Cell Growth Quantification for MytiLec-1 Treated Cells
		3.3 Lectin-Dependent Signal Transduction
	4 Notes
	References
Chapter 22: Lectin-Type Ubiquitin Ligase Subunits: Fbs Proteins and Their Applications for Use
	1 Introduction
	2 Materials
		2.1 N-Glycopeptide Enrichment Method
		2.2 Detecting Damaged Organelles in Cells Using Fbs3
	3 Methods
		3.1 N-Glycopeptide Enrichment Method
			3.1.1 Preparation of Recombinant Fbs1 GYR Protein
			3.1.2 Preparation of Sample Peptide from Cultured Cells
			3.1.3 Enrichment of N-Glycopeptides by N-Glyco-FASP Method
		3.2 Detection of Damaged Organelles in Cells by Using Fbs3
			3.2.1 Plasmid Construction and Transfection
			3.2.2 Detecting Organelle Damage by Time-Lapse Imaging
	4 Notes
	References
Chapter 23: F-Type Lectins: Structure, Function, and Evolution
	Abbreviations
	1 Introduction
	2 Structural Features of FTLs
		2.1 The FTL Structural Fold
		2.2 Carbohydrate Recognition by the FTLD
		2.3 Domain and Subunit Organization of FTLs
			2.3.1 FTLs with Tandemly Arrayed FTLDs
			2.3.2 FTL Isoforms and Diversity in Ligand Recognition
			2.3.3 Oligomeric Organization of FTL Subunits
			2.3.4 Structural Diversification of FTLs
	3 Taxonomic Distribution and Evolution of the FTLD
	4 Biological Roles of FTLs
	5 Conclusions
	References
Chapter 24: Purification and Biochemical Characterization of Selected F-Type Lectins
	Abbreviations
	1 Introduction
	2 Materials
		2.1 Purification of FTLs
		2.2 Biochemical Characterization of FTLs
	3 Methods
		3.1 Preparation of Liver Tissue Extracts
		3.2 Preparation of Serum
		3.3 Expression of Recombinant FTL Proteins
		3.4 FTL Isolation by Affinity Chromatography
		3.5 FTL Purification by Size Exclusion Chromatography (SEC)
		3.6 Edman Sequence Analysis
		3.7 Assessment of the Native Molecular Size by SEC
		3.8 ELISA for Analysis of Carbohydrate-Binding Activity and Specificity
		3.9 Agglutination Assay
		3.10 Agglutination-Inhibition Assay
		3.11 Glycan Array
	4 Notes
	References
Chapter 25: LecA (PA-IL): A Galactose-Binding Lectin from Pseudomonas aeruginosa
	1 Introduction
	2 Materials
		2.1 Gene Sequence
		2.2 Protein Production in Escherichia coli
		2.3 Protein Purification
		2.4 Surface Plasmon Resonance
		2.5 Protein Crystallization
	3 Methods
		3.1 LecA Production
			3.1.1 Transformation and Preculture
			3.1.2 Culture
			3.1.3 LecA Purification by Affinity Chromatography
		3.2 Surface Plasmon Resonance (SPR)
			3.2.1 Sample Preparation
			3.2.2 LecA Immobilization on SPR Chip Using Amine Coupling Method
		3.3 LecA Crystallization
			3.3.1 Sample Preparation
			3.3.2 Hanging Drop Vapour Diffusion
	4 Notes
	References
Chapter 26: Glycan Recognition and Application of P-Type Lectins
	1 Introduction
	2 Materials
		2.1 Preparation of Recombinant Proteins
		2.2 SDS-PAGE
		2.3 Lectin Blotting
	3 Methods
		3.1 Expression and Purification of Recombinant Human Dom9-his by Methylotrophic Yeast
		3.2 Lectin Blotting with the Dom9-his as a Probe
	4 Notes
	References
Chapter 27: Purification and Assays of Tachylectin-5
	1 Introduction
	2 Materials
		2.1 Preparation of Hemolymph Plasma
		2.2 Purification of Tachylectin-5
			2.2.1 Preparation of Acetyl Group-Immobilized Resin (Acetyl-Toyopearl)
			2.2.2 Acetyl-Toyopearl Column Chromatography
		2.3 Hemagglutination Assay
		2.4 Bacterial Agglutination Assay
		2.5 A Synergistic Effect of Tachylectin-5A or Tachylectin-5B on Antimicrobial Activity of Big Defensin
	3 Methods
		3.1 Preparation of Hemolymph Plasma
		3.2 Purification of Tachylectin-5
			3.2.1 Preparation of Acetyl-Toyopearl
			3.2.2 Acetyl-Toyopearl Column Chromatography
		3.3 Hemagglutination Assay
		3.4 Bacterial Agglutination Assay
		3.5 A Synergistic Effect of Tachylectin-5A or Tachylectin-5B on Antimicrobial Activity of Big Defensin
	4 Notes
	References
Chapter 28: Preparation of Soluble Malectin and Its Tetramer
	1 Introduction
	2 Materials
		2.1 Construction of Plasmids for sMAL Expression in Escherichia coli
		2.2 Expression and Purification of sMAL
		2.3 Biotinylation of sMAL and Tetramer Preparation for Cell Binding Assay
	3 Methods
		3.1 Construction of Plasmids for sMAL Expression in E. coli
		3.2 Expression and Purification of sMAL
		3.3 Biotinylation of sMAL and Tetramer Preparation for Cell Binding Assay
	4 Notes
	References
Chapter 29: Calnexin/Calreticulin and Assays Related to N-Glycoprotein Folding In Vitro
	1 Introduction
	2 Materials
		2.1 Expression of CRT in Bacteria
		2.2 Purification of CRT
			2.2.1 Glutathione Sepharose Column Chromatography
			2.2.2 Cleavage of GST-CRT Fusion Protein
			2.2.3 Mono Q Anion-Exchange Liquid Chromatography
		2.3 Aggregation Suppression Assay
		2.4 Heat-Induced Inactivation of Jack Bean α-Mannosidase
		2.5 The Enzyme Assay for α-Mannosidase
	3 Methods
		3.1 Expression of Mammalian CRT in Bacteria
		3.2 Purification of Mammalian CRT Expressed in Bacteria
			3.2.1 Glutathione Sepharose Column Chromatography
			3.2.2 Cleavage of GST-CRT Fusion Protein
			3.2.3 Mono Q Anion-Exchange Chromatography
		3.3 Aggregation Suppression Assay
		3.4 Heat-Induced Inactivation of Jack Bean α-Mannosidase
		3.5 The Enzyme Assay for α-Mannosidase
	4 Notes
	References
Chapter 30: Purification and Assays of Tachylectin-2
	1 Introduction
	2 Materials
		2.1 Preparation of Hemocyte Extract
		2.2 Purification of Tachylectin-2
			2.2.1 Preparation of Dextran Sulfate-Sepharose CL-6B
			2.2.2 Dextran Sulfate-Sepharose CL-6B Column Chromatography
			2.2.3 CM-Sepharose CL-6B Column Chromatography
			2.2.4 Mono S Column Chromatography
		2.3 Hemagglutination Assay
		2.4 Bacterial Agglutination Assay
		2.5 Determination of Association Constants
		2.6 Analytical Ultracentrifugation
	3 Methods
		3.1 Preparation of Hemocyte Extract
		3.2 Purification of Tachylectin-2
			3.2.1 Preparation of Dextran Sulfate-Sepharose CL-6B
			3.2.2 Dextran Sulfate-Sepharose CL-6B Column Chromatography
			3.2.3 CM-Sepharose CL-6B Column Chromatography
			3.2.4 Mono S Column Chromatography
		3.3 Hemagglutination Assay
		3.4 Bacterial Agglutination Assay
		3.5 Determination of Association Constants
		3.6 Analytical Ultracentrifugation
	4 Notes
	References
Chapter 31: Purification and Assays of Tachycitin
	1 Introduction
	2 Material
		2.1 Preparation of Hemocyte Debris
		2.2 Purification of Tachycitin
			2.2.1 Preparation of Protein Extract from Hemocyte Debris
			2.2.2 Sephadex G-50 Column Chromatography
			2.2.3 S-Sepharose FF Column Chromatography
		2.3 Antimicrobial Activity
		2.4 Bacterial Agglutination Assay
		2.5 Analytical Ultracentrifugation
	3 Methods
		3.1 Preparation of Hemocyte Debris
		3.2 Purification of Tachycitin
			3.2.1 Preparation of Protein Extract from Hemocyte Debris
			3.2.2 Sephadex G-50 Column Chromatography
			3.2.3 S-Sepharose FF Column Chromatography
		3.3 Antimicrobial Activity
		3.4 Bacterial Agglutination Assay
		3.5 Analytical Ultracentrifugation
	4 Notes
	References
Chapter 32: Methods for Purifying Datura stramonium Agglutinin and Producing Recombinant Agglutinin Protein in a Heterologous ...
	1 Introduction
	2 Materials
		2.1 Plant Materials
		2.2 Bacterial Strains
		2.3 cDNA Material
		2.4 Hemagglutination Assay
		2.5 Preparation of Chitin-Gel Via Chitosan Acetylation
		2.6 Buffers and Materials for Purifications of Native and Recombinant DSA
		2.7 Buffers and Media for Transformation of Arabidopsis thaliana
		2.8 Detection of Transgene and Recombinant Protein
	3 Methods
		3.1 Hemagglutination Assay
		3.2 Preparation of Chitin-Gel Via Chitosan Acetylation
		3.3 Purification of DSA Isolectins from Datura Seeds Using Affinity Chromatography
		3.4 Separation of DSA Isolectins Using Hydrophobic Interaction Chromatography
		3.5 Generation of DSA-B Transgenic Arabidopsis Plants
		3.6 Purification of DSA Recombinant BB-Isolectin from Transgenic Arabidopsis Plants
	4 Notes
	References
Chapter 33: ZG16p, an Animal Homologue of Plant β-Prism Fold Lectins: Purification Methods of Natural and Recombinant ZG16p an...
	1 Introduction
	2 Materials
		2.1 Purification of ZG16p from Rat Pancreas (Rat ZG16p)
		2.2 Expression and Purification of Recombinant ZG16p
		2.3 Inhibition of Cancer Cell Growth by ZG16p
			2.3.1 Cell Culture
			2.3.2 ZG16p Solution
			2.3.3 Cell Proliferation Assay
	3 Methods
		3.1 Purification of Rat ZG16p
			3.1.1 Preparation of Zymogen Granule Extracts
			3.1.2 Purification of Rat ZG16p Using Heparin Column Chromatography
		3.2 Expression and Purification of Recombinant ZG16p
		3.3 Inhibition of Cancer Cell Growth by ZG16p
	4 Notes
	References
Chapter 34: ArtinM: Purification and Evaluation of Biological Activities
	1 Introduction
	2 Materials
		2.1 Purification of ArtinM
		2.2 Analysis of the Purity of ArtinM
		2.3 Endotoxin Removal from ArtinM Solution
		2.4 Biological Activity of ArtinM
	3 Methods
		3.1 Preparation of Jackfruit Crude Seed Extract
		3.2 Purification of ArtinM
			3.2.1 Jacalin Removal from Jackfruit Crude Seed Extract
			3.2.2 Purification of ArtinM from Crude Seed Extract Depleted of Jacalin
		3.3 Endotoxin Removal from ArtinM Solution
		3.4 Biological Activity of ArtinM
	4 Notes
	References
Chapter 35: Bacterial Expression of Rhamnose-Binding Lectin from Catfish Eggs
	1 Introduction
	2 Materials
		2.1 Protein Expression in KRX and Isolation of Inclusion Bodies
		2.2 Denaturation, Refolding, and Purification
			2.2.1 Buffers
			2.2.2 Affinity Resins and the Others
		2.3 Protein Detection
		2.4 Hemagglutination and Inhibition Assays
	3 Methods
		3.1 Protein Expression in KRX and Isolation of Inclusion Bodies
		3.2 Denaturation, Refolding, and Purification of Recombinants Except for D3DeltaC and D3DeltaN (See Note 2)
		3.3 Denaturation, Purification, and Refolding of D3DeltaC and D3DeltaN
		3.4 Protein Detection
		3.5 Hemagglutination and Inhibition Assays
	4 Notes
	References
Chapter 36: A Bioassay for Determining Symbiotic Zooxanthellae Shape Control Using Lectin SLL-2 from the Octocoral Sinularia l...
	1 Introduction
	2 Materials
		2.1 Selecting Zooxanthellae
		2.2 Zooxanthellae
		2.3 Medium
		2.4 Cultivating and Observing Zooxanthellae
		2.5 Preparation of Carbohydrate-Fixed Resin
		2.6 Lectin Preparation
	3 Methods
		3.1 Selecting Zooxanthellae
		3.2 Preparation of Sterilised Medium
		3.3 Cultivating Zooxanthellae and Plotting Growth Curve
		3.4 Measuring Diurnal Change in Zooxanthella Form
		3.5 Counting the Number of Zooxanthellae Using Fluorescence
		3.6 Preparation of Galactose-Fixed Resin
		3.7 Preparing a Lectin SLL-2 from the Coral Sinularia Lochmodes
		3.8 Bioassay for Symbiosis-Related Factor
	4 Notes
	References
Chapter 37: MIC4 from Toxoplasma gondii: A Lectin Acting as a Toll-Like Receptor Agonist
	1 Introduction
	2 Material
		2.1 Plasmid Constructs
		2.2 Cells
		2.3 Culture Medium
		2.4 Buffers and Solutions
		2.5 Animal
		2.6 Agonists of TLR
		2.7 Others
	3 Methods
		3.1 Expression of Recombinant MIC4 (rMIC4) and rMIC4(K469M)
		3.2 rMIC4 and rMIC4(K469M) SDS-PAGE
		3.3 Isolation of Inclusion Bodies Through Centrifugation
		3.4 Inclusion Bodies Solubilization
		3.5 Ni-Sepharose Chromatography
		3.6 Protein Refolding by Dialysis Method
		3.7 Obtention, Culture, and Phenotyping of Bone Marrow-Derived Macrophage (BMDM)
		3.8 The rMIC4 and rMIC4(K469M) Activity on Macrophages
		3.9 The rMIC4 Activity on TLR2-Transfected HEK293T Cell Line: A Gene Reporter Assay
	4 Notes
	References
Chapter 38: Production and Characterization of MIC1: A Lectin from Toxoplasma gondii
	1 Introduction
	2 Materials
		2.1 The Expression Plasmid
		2.2 Competent Cells for Chemical Transformation
		2.3 Culture Medium
		2.4 Buffers and Solutions
		2.5 Antibodies
		2.6 Others
	3 Methods
		3.1 Expression of Recombinant MIC1 (rMIC1) and rMIC1 (T126A/T220A)
		3.2 rMIC1 and rMIC1(T126A/T220A) SDS-PAGE
		3.3 Isolation of Inclusion Bodies Through Centrifugation
		3.4 Inclusion Body Solubilization
		3.5 Ni-Sepharose Chromatography
		3.6 Protein Refolding by Dialysis Method
		3.7 Sugar-Binding Assay
	4 Notes
	References
Chapter 39: Affinity Labeling and Purification of Plant Chitin-Binding LysM Receptor with Chitin Octasaccharide Derivatives
	1 Introduction
	2 Materials
		2.1 Preparation of Microsomal Membrane Fraction
		2.2 Preparation of Biotinylated Chitooligosaccharide
		2.3 Affinity Biotinylation
		2.4 Purification of CEBiP
		2.5 Equipment
	3 Methods
		3.1 Preparation of Microsomal Membrane Fraction from Rice Cultured Cells
		3.2 Preparation of a Biotinylated Chitooligosaccharide
		3.3 Affinity Labeling of a LysM Protein CEBiP in Rice Microsomal Membrane Fraction
		3.4 Preparation of Affinity Columns for the Purification
		3.5 Purification of CEBiP from Rice Plasma Membrane
	4 Notes
	References
Chapter 40: Purification of GNA-Related Lectins from Natural Sources
	Abbreviations
	1 Introduction
	2 Materials
	3 Methods
		3.1 Lectin Extraction
		3.2 Lectin Purification
			3.2.1 Ion Exchange Chromatography
			3.2.2 Affinity Chromatography on Mannose-Sepharose 4B
	4 Notes
	References
Chapter 41: Expression, Purification, and Applications of the Recombinant Lectin PVL from Psathyrella velutina Specific for Te...
	1 Introduction
	2 Materials
		2.1 Production of rPVL
		2.2 Purification of rPVL
		2.3 Hemagglutination Assays (HA)
		2.4 Affinity Measurements by Surface Plasmon Resonance (SPR)
		2.5 rPVL Labelling
		2.6 Enzyme-Linked Lectin Assays (ELLA)
		2.7 Western Blotting of O-GlcNAcylated Proteins
		2.8 Immunofluorescence Analysis on Whole Cells
		2.9 Immunohistochemistry
	3 Methods
		3.1 Production of rPVL
		3.2 Purification of rPVL
		3.3 Hemagglutination Assays (HA)
		3.4 Affinity Measurements by SPR
		3.5 rPVL Labelling
			3.5.1 Biotinylation
			3.5.2 Horseradish Peroxidase Coupling
			3.5.3 Alexa Fluor 488 Coupling
		3.6 Enzyme-Linked Lectin Assay
		3.7 Western Blotting of O-GlcNAcylated Proteins
		3.8 Immunofluorescence Analysis on Whole Cells
		3.9 Immunohistochemistry
	4 Notes
	References
Chapter 42: Yeast Flocculin: Methods for Quantitative Analysis of Flocculation in Yeast Cells
	1 Introduction
	2 Materials
		2.1 Stock Solutions
		2.2 Flocculation Assay
	3 Method
		3.1 Flocculation Assay
	4 Notes
	References
Chapter 43: Pleurotus cornucopiae Mycelial Lectin (PCL-M): Purification and Detection of the Activity on Mycelial Surface
	1 Introduction
	2 Materials
		2.1 Organism
		2.2 Culture Medium
		2.3 Hemagglutination Assay
		2.4 Purification
	3 Methods
		3.1 Culturing of the Mycelia
		3.2 Assay Procedure
		3.3 Purification of PCL-M
		3.4 Observation of Adhesion of Red Blood Cells to Mycelia Mediated by PCL-M
	4 Notes
	References
Chapter 44: Expression and Purification of a Human Pluripotent Stem Cell-Specific Lectin Probe, rBC2LCN
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Instruments
	3 Methods
		3.1 Construction of rBC2LCN-pET27b Plasmid
		3.2 Expression of rBC2LCN-pET27b Plasmid in E. coli
		3.3 Preparation of l-Fucose-Sepharose for Affinity Purification of rBC2LCN
		3.4 Affinity Purification
		3.5 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) of Eluted Fractions
		3.6 Dialysis of Eluted Fractions
		3.7 Protein Quantification and Preservation
		3.8 Cy3 Labeling of rBC2LCN
		3.9 Live Staining
	4 Notes
	References
Chapter 45: Preparation of Fluorescent Recombinant Shiga Toxin B Subunit and Its Application to Flow Cytometry
	1 Introduction
	2 Materials
		2.1 Expression of Stx1B in E. coli
		2.2 Purification of Stx1B
		2.3 Fluorescent Labeling
		2.4 Flow Cytometry
	3 Methods
		3.1 Purification of Recombinant Stx1B
		3.2 Fluorescence Conjugation of Recombinant Stx1B
		3.3 Application of Fluorescent Stx1B to Flow Cytometry
	4 Notes
	References
Chapter 46: LecB, a High Affinity Soluble Fucose-Binding Lectin from Pseudomonas aeruginosa
	1 Introduction
	2 Materials
		2.1 LecB Production
		2.2 LecB Purification
		2.3 Quality Control by Evaluation of Affinity
	3 Methods
		3.1 LecB Production
			3.1.1 Transformation and Preculture
			3.1.2 Culture
		3.2 LecB Purification
		3.3 Quality Control by Evaluation of Affinity
			3.3.1 Sample Preparation
			3.3.2 ITC Experiment
	4 Notes
	References
Chapter 47: Sialoglycovirology of Lectins: Sialyl Glycan Binding of Enveloped and Non-enveloped Viruses
	1 Introduction
	2 Determination of Sia-Binding Specificities for Viruses
	3 Sialylglycoconjugates as Host Receptor Determinants of Viral Spike Glycoproteins for Enveloped Viral Infection
		3.1 Orthomyxoviridae
		3.2 Paramyxoviridae
		3.3 Coronaviridae
	4 Sialylglycoconjugates as Host Receptor Determinants of Viral Capsid Proteins for Non-enveloped Viral Infection
		4.1 Picornaviridae
		4.2 Caliciviridae
		4.3 Reoviridae
		4.4 Adenoviridae
		4.5 Parvoviridae
		4.6 Polyomaviridae
	5 Molecular and Structural Basis of Viral Lectin-Sialyl Glycan Interactions
	6 Conclusions, Perspectives, and Future Directions
	References
Chapter 48: Hemagglutinin Inhibitors are Potential Future Anti-Influenza Drugs for Mono- and Combination Therapies
	1 Introduction
	2 Hemagglutinin Inhibitors (HAIs) of Influenza A Viruses
		2.1 Mode of Terminal Sialyl-Galactose Linkage of Sialyl Sugar Chain Receptors That Bind to Influenza A Virus HA
		2.2 Molecular Length and Structure of Sialyl Glycan Receptors
		2.3 Multivalent Sialyl Sugar Chain Dimension That Can Reach the Binding Site of HA
		2.4 Clustering of Sialyl Sugar Chains on Macromolecular Scaffolds
		2.5 Modification of the C-3 Position of Terminal Sialic Acid in Sialyl Glycan Inhibits Catalytic Hydrolysis of Influenza Virus...
	3 Synergizing the HAI Activity by Influenza Virus Neuraminidase-Targeting Drugs
	4 Conclusion
	References
Chapter 49: Preparation and Detection of Glycan-Binding Activity of Influenza Virus
	1 Introduction
	2 Materials
		2.1 Propagation of Influenza Viruses
			2.1.1 General Materials
			2.1.2 Amplification of Influenza Virus in Embryonated Chicken Eggs
			2.1.3 Amplification of Swine and Human Influenza Virus in MDCK Cells
		2.2 Determination of Their Hemagglutination Units
		2.3 Preparation of Glycan Array for Influenza Virus Detection
		2.4 Detection of Influenza Virus Using Glycan Array
	3 Methods
		3.1 Propagation of Influenza Viruses
			3.1.1 Amplification of Avian Influenza Virus in Embryonated Chicken Eggs
			3.1.2 Amplification of Classical Swine and Human Influenza Viruses in MDCK Cells
		3.2 Relative Quantities of Virus Particles by Hemagglutination Assay
			3.2.1 Preparation of 0.5% Erythrocyte Suspension in PBS
			3.2.2 Determination of Hemagglutination Units
		3.3 Preparation of Glycan Array for Influenza Virus Detection
			3.3.1 Conversion of SGP to α2,3SGP
			3.3.2 Preparation of the Neoglycoprotein
		3.4 Binding Assay Using the Neoglycoprotein
	4 Notes
	References
Chapter 50: Screening for Components/Compounds with Anti-Rotavirus Activity: Detection of Interaction Between Viral Spike Prot...
	1 Introduction
	2 Materials
		2.1 MA104 Cell Culture
		2.2 Virus Purification
		2.3 Screening of Rotavirus-Binding Glycoproteins
		2.4 Infection Assay and Immunofluorescence Detection
	3 Methods
		3.1 Rotavirus Purification
		3.2 Screening of Rotavirus-Binding Glycoproteins by Using Evanescent Field-Based Fluorescence-Assisted Detection
			3.2.1 Trypsin Activation
			3.2.2 Rotavirus-Glycoprotein-Binding Assay
		3.3 Rotavirus Infection Assay Using MA104 Cells
			3.3.1 MA104 Cell Maintenance
			3.3.2 Cell Preparation for Infection Assay
			3.3.3 Neutralization Assay
			3.3.4 Pretreatment Assay
			3.3.5 Indirect Immunofluorescence Detection
	4 Notes
	References
Chapter 51: ELISA-Based Methods to Detect and Quantify Norovirus Virus-Like Particle Attachment to Histo-Blood Group Antigens
	1 Introduction
	2 Materials
		2.1 Expression and Purification of Recombinant VLPs
		2.2 Hemagglutination Inhibition Assay
		2.3 Saliva-VLP Binding Assay
		2.4 Synthetic Carbohydrate-VLP Binding Assay
	3 Methods
		3.1 Expression and Purification of Recombinant VLPs
		3.2 Detection of Soluble ABH Antigens in Saliva by Hemagglutination Inhibition Assay
		3.3 ELISA-Based Binding Assay 1: Saliva-VLP Binding Assay
		3.4 ELISA-Based Binding Assay 2: Synthetic Carbohydrate-VLP Binding Assay
	4 Notes
	References
Chapter 52: FAM3B/PANDER-Like Carbohydrate-Binding Domain in a Glycosyltransferase, POMGNT1
	1 Introduction
	2 Materials
		2.1 Lectin-Like Activity of POMGNT1 Stem Domain
			2.1.1 Preparation of Recombinant Stem Domain
			2.1.2 Carbohydrate-Binding Assay of Stem Domain
		2.2 Functional Assay of Stem Domain
			2.2.1 Cell Culture, Transfection, and Preparation of Cell Membrane Fraction
			2.2.2 Enrichment of α-DG and Western Blotting
			2.2.3 Glycosyltransferase Assay
	3 Methods
		3.1 Lectin-Like Activity of POMGNT1 Stem Domain
			3.1.1 Preparation of Recombinant POMGNT1stem
			3.1.2 Carbohydrate-Binding Assay of POMGNT1stem
		3.2 Functional Assay of Stem Domain by Rescue Experiment in IIH6 Epitope Synthesis
			3.2.1 Cell Culture, Transfection, and Preparation of Cell Membrane Fraction
			3.2.2 Enrichment of α-DG and Western Blotting
			3.2.3 Glycosyltransferase Assay of POMGNT1
	4 Notes
	References
Chapter 53: Mannose-Specific Oyster Lectin CGL1
	1 Introduction
	2 Materials
		2.1 Mannose-Immobilized Resin
			2.1.1 Reagents/Solutions
			2.1.2 Equipment
		2.2 Purification of CGL1
			2.2.1 Reagents/Solutions/Materials
			2.2.2 Equipment
	3 Methods
		3.1 Preparation of the Mannose-Immobilized Resin
		3.2 Purification of CGL1
	4 Notes
	References
Chapter 54: Receptor-Binding Assays of Enterovirus D68
	1 Introduction
	2 Materials
		2.1 Regents
		2.2 Antibodies
		2.3 Cells
		2.4 Experimental Animals
		2.5 Equipment
	3 Methods
		3.1 Clinical Sample Handling
		3.2 Virus Isolation
		3.3 Preparation of Polyclonal Antibody
		3.4 Preparation of Slides for Glycan Array Analysis
		3.5 Modification of Erythrocytes
		3.6 Glycan Array Analysis
		3.7 Hemagglutination Test Using Modified Erythrocytes
	4 Notes
	References
Chapter 55: Large-Scale Expression and Purification of Mumps Virus Hemagglutinin-Neuraminidase for Structural Analyses and Gly...
	1 Introduction
	2 Materials
		2.1 Large-Scale MuV-HN Protein Expression
		2.2 Purification and Modification of His-Tagged MuV-HN Proteins
		2.3 Crystallization of the MuV-HN Protein
	3 Methods
		3.1 Large-Scale MuV-HN Protein Expression
			3.1.1 Preparation of Expression Vectors Encoding MuV-HN Proteins
			3.1.2 Large-Scale Cell Culture
			3.1.3 Transient Transfection of HEK293S GnTI(-) Cells with Expression Vectors
		3.2 Purification and Modification of the His-Tagged MuV-HN Protein
			3.2.1 Affinity Chromatography
			3.2.2 Methylation of Lysine Residues
			3.2.3 Size Exclusion Chromatography (SEC)
		3.3 Crystallization of the MuV-HN Protein
	4 Notes
	References
Chapter 56: Purification of AJLec: A Novel Galactose-Specific Lectin from the Sea Anemone Anthopleura japonica
	1 Introduction
	2 Materials
		2.1 Lactose-Immobilized Resin
			2.1.1 Reagents/Solutions
			2.1.2 Equipment
		2.2 Purification of AJLec
			2.2.1 Reagents/Solutions/Materials
			2.2.2 Equipment
	3 Methods
		3.1 Preparation of the Lactose Resin
		3.2 Purification of AJLec
	4 Notes
	References
Chapter 57: Annexin Lectins: Ca2+-Dependent Heparin-Binding Activity, Phosphatidylserine-Binding Activity, and Anticoagulant A...
	1 Introduction
	2 Materials
		2.1 Expression and Purification of GST (Glutathione S-Transferase)-Tagged Annexins (GST-ANXs)
		2.2 Solid-Phase Heparin-Binding Assay
		2.3 Solid-Phase PS-Binding Assay
		2.4 Inhibition Assay of Plasma Coagulation Reaction
	3 Methods
		3.1 Expression and Purification of GST-ANXs
		3.2 Solid-Phase Heparin-Binding Assay
			3.2.1 Preparation of BSA-Heparin
			3.2.2 Binding Assay
		3.3 Solid-Phase PS-Binding Assay
		3.4 Inhibition Assay of Plasma Coagulation Reaction
	4 Notes
	References
Chapter 58: Expression, Purification, and Functional Characterization of Tectonin 2 from Laccaria bicolor: A Six-Bladed Beta-P...
	1 Introduction
	2 Materials
		2.1 Production of Recombinant Lb-Tec2
		2.2 Purification of Recombinant Lb-Tec2
		2.3 Isothermal Titration Microcalorimetry
		2.4 Surface Plasmon Resonance
		2.5 Caenorhabditis elegans Biotoxicity Assay
		2.6 Localization of Lb-Tec2-Binding in C. elegans
		2.7 Lectin Blot
		2.8 Bacterial Agglutination Assay
	3 Methods
		3.1 Production of Recombinant Lb-Tec2
		3.2 Purification of Recombinant Lb-Tec2
		3.3 Isothermal Titration Microcalorimetry
		3.4 Surface Plasmon Resonance
		3.5 C. elegans Biotoxicity Assay
		3.6 Localization of Lb-Tec2-binding in C. elegans
		3.7 Lectin Blot
		3.8 Bacterial Agglutination Assay
			3.8.1 Assay with Purified Protein
			3.8.2 Assay with Soluble Protein Extract
	4 Notes
	References
Chapter 59: The OAAH Family: Anti-Influenza Virus Lectins
	1 Introduction
	2 Materials
		2.1 Cyanobacterium
		2.2 Chromatography
		2.3 Virus
		2.4 Host Cell
		2.5 Amide Black Assay
		2.6 Neutral Red Dye Uptake Assay
		2.7 Immunofluorescence Microscopy
		2.8 Surface Plasmon Resonance Analysis
		2.9 Enzyme-Linked Immunosorbent Assay (ELISA)
	3 Methods
		3.1 Purification of the Lectin (OAA)
		3.2 Assay for Anti-Influenza Activity of Lectins
			3.2.1 Amide Black Assay
			3.2.2 Neutral Red Dye Uptake Assay
			3.2.3 Direct Immunofluorescence Microscopy
		3.3 Assay for Interaction Between Lectin and Influenza Viral Hemagglutinin (HA)
			3.3.1 Surface Plasmon Resonance (SPR) Analysis with a Biacore X100
			3.3.2 ELISA Assay
	4 Notes
	References
Correction to: Hemagglutinin Inhibitors are Potential Future Anti-Influenza Drugs for Mono- and Combination Therapies
Index