Chemical glycobiology. Part A, Synthesis, manipulation and applications of glycans / edited by Barbara Imperiali.

Format:

Book

Contributor:

Imperiali, Barbara, editor.

Series:

Methods in enzymology ; Volume 599.

Methods in Enzymology, 0076-6879 ; Volume 599

Language:

English

Subjects (All):

Glycoconjugates.

Glycomics.

Physical Description:

1 online resource (432 pages) : illustrations (some color).

Place of Publication:

Cambridge, Massachusetts : Academic Press, 2017.

Summary:

Chemical Glycobiology, Volume 597, the latest release in the Methods in Enzymology series, continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume, the first on chemical glycobiology, contains comprehensive chapters on the Discovery of New Glycosidases from Metagenomic Libraries, Structure-guided directed evolution of glycosidases: A case study in engineering a blood group antigen-cleaving enzyme, A Pipeline for Studying and Engineering Single-Subunit Oligosaccharyltransferases, Directed evolution of glycopeptides using mRNA display, Chemoenzymatic Synthesis and Applications of Prokaryote-Specific UDP-Sugars, and Biosynthesis of Legionaminic Acid and its Incorporation into Glycoconjugates.Readers will find the latest information on this developing area of research, as reported by leaders in the field.- Presents an updated volume in this regular series- Covers research on chemical glycobiology

Contents:

Front Cover

Chemical Glycobiology Part A. Synthesis, Manipulation and Applications of Glycans

Copyright

Contents

Contributors

Preface

References

Section I: Evolution and Engineering of Glycans and Glycan Processing Enzymes

Chapter One: Discovery of New Glycosidases From Metagenomic Libraries

1. Introduction

2. DNA Isolation and Purification

2.1. Indirect DNA Isolation

2.2. Direct DNA Isolation

2.2.1. Equipment

2.2.2. Buffers and Reagents

2.2.3. Protocol

2.3. DNA Purification

3. Library Generation

3.1. Large Insert Fosmid Libraries

3.1.1. Equipment

3.1.2. Buffers and Reagents

3.1.3. Protocol

3.1.3.1. DNA End Repair

3.1.3.2. Size Selection

3.1.3.3. Ligation

3.1.3.4. Phage Packaging and Transduction

3.2. Small Insert Library

3.2.1. Pipette Tip Shearing of DNA

3.2.2. Enzymatic DNA Digestion

3.2.3. Ligation and Transformation

4. Library Screening

4.1. Equipment

4.2. Buffers and Reagents

4.3. Protocol

5. Preparation for Sequencing

5.1. Fosmid Isolation

5.1.1. Equipment

5.1.2. Buffers and Reagents

5.1.3. Protocol

6. Concluding Remarks

Acknowledgments

Chapter Two: Structure-Guided Directed Evolution of Glycosidases: A Case Study in Engineering a Blood Group Antigen-Cleav ...

2. Structure-Guided Library Design and Generation

2.1. Identification of Amino Acid Target Residues From a Glycosidase Structure

2.2. Generating Glycosidase Mutant Libraries Through Site-Saturation Mutagenesis (by Overlap Extension)

2.2.2. Reagents and Buffers

2.2.3. Procedure

2.2.4. Notes

2.3. Generating Glycosidase Mutant Libraries Through Error-Prone PCR

2.3.1. Equipment

2.3.2. Reagents and Buffers

2.3.3. Procedure

2.3.4. Notes.

3. Enzymatic Preparation of Fluorogenic Oligosaccharides as Substrates for High-Throughput Assays

3.1. In Vitro Enzymatic Synthesis of Fluorogenic Oligosaccharides

3.1.2. Buffers and Solutions

3.1.3. Procedure

3.1.4. Notes

3.2. Biosynthesis of Fluorogenic Oligosaccharides in Metabolically Engineered Bacteria

4. Performing Enzymatic Assays

4.1. High-Throughput Assays of Glycosidase Variants Expressed From Mutant Libraries

4.1.1. Equipment

4.1.2. Buffers, Solutions, and Media

4.1.3. Procedure

4.1.4. Notes

4.2. Determining the Enzyme Kinetics of Mutant Glycosidases

4.2.1. Equipment and Software Tools

4.2.2. Buffers, Solutions, and Reagents

4.2.3. Procedure

4.2.4. Notes

5. Summary and Conclusion

Chapter Three: A Pipeline for Studying and Engineering Single-Subunit Oligosaccharyltransferases

2. Materials

2.1. Media

2.2. Media Supplements

2.3. Bacterial Strains and Plasmids

2.4. GlycoSNAP Assay

2.5. Preparation of ssOSTs by CFPS

2.5.1. S30 Extract Preparation

2.5.2. Producing ssOST in CFPS Supplemented With POPC Nanodiscs

2.6. Protein Purification and Crude Membrane Extracts Containing ssOST or LLOs

2.7. Extraction of LLOs

2.8. IVG Reaction

2.9. Lectin Blot and Western Blot Analysis of Glycosylation Products

3. Methods

3.1. GlycoSNAP Assay

3.2. Preparation of ssOST by CFPS

3.2.1. S30 Extract Preparation

3.2.2. Producing ssOST in CFPS Supplied With POPC Nanodiscs

3.3. Preparation of Purified and Crude Membrane Extract Glycosylation Components

3.3.1. Preparation of Crude Membrane Extract or Purified ssOST Enzyme

3.3.2. Purification of Acceptor Protein scFv13-R4DQNAT

3.3.3. Extraction of LLOs Bearing C. jejuni Glycan (Adapted From Guarino & DeLisa, 2012)

3.4. IVG Setup.

3.5. Detection of Glycoprotein From In Vivo and IVG Assay

4. Conclusion

5. Notes

Chapter Four: Directed Evolution of Glycopeptides Using mRNA Display

2. Overview of the PURE System

3. Quality Control/Evaluation of Target Protein

3.1. Confirmation of Target Activity

3.2. Confirmation of Target Capture Method

3.3. Choosing a Method for the Retrieval of cDNA From Selected Fusions

4. Glycopeptide mRNA Display Selection Procedures

4.1. Design and Preparation of Library DNA

4.1.1. Purification of the Library DNA

4.1.2. Optional Procedure: TA Cloning of DNA Libraries and Sequencing

4.2. Library RNA Preparation by T7 Transcription

4.2.1. T7 Transcription Using a Kit and Synthetic DNA

4.2.2. Alternative Protocol for T7 Transcription

4.3. Modification of RNA With Puromycin

4.3.1. Optional Procedure: Purification of Puromycin Modified RNA

4.4. Translation and Formation of RNA-Peptide Fusions

4.5. Purification of Fusions

4.5.1. Oligo(dT) Purification

4.5.2. Fusion Purification Using Oligo(dT) Cellulose

4.5.3. Alternative Fusion Purification Using Oligo(dT) Magnetic Beads

4.5.4. Ni-NTA Agarose Purification

4.5.5. Desalting by Gel Filtration

4.6. Reverse Transcription to Form cDNA/RNA-Peptide Fusions

4.7. Click Glycosylation

4.7.1. Alternative Protocol for Click Glycosylation

4.7.2. Evaluation of Click Reaction

4.8. First Round Selection

4.9. PCR Amplification of the DNA of Selected Fusions

4.10. Subsequent Rounds of Selections

4.11. Determination of Selected Sequences

5. Binding Assay Utilizing Translated Glycopeptides

5.1. Preparation of Radioactive Glycopeptides

5.2. Measurement of Binding Constants by Radiometric Bead-Binding Assay

6. Discussion, Summary, and Conclusions

Appendix.

A.1. RNA Precautions

A.2. Preparation of PURE System Components

A.2.1. Preparation of His6-Tagged Proteins

A.2.2. Preparation of Ribosomes

A.2.3. Other PURE System Components

Section II: Chemical and Enzymatic Synthesis and Analysis of Glycans and Glycoconjugates

Chapter Five: Chemoenzymatic Synthesis and Applications of Prokaryote-Specific UDP-Sugars

2. Equipment and Consumables

3. Chemoenzymatic Preparation of UDP-diNAcBac With PglF, PglC, and PglD

3.1. Discussion

3.2. Procedure

3.2.1. Expression of PglF (GST-PglFΔ1-130) From C. jejuni, PglC From N. gonorrhoeae, and PglD From C. jejuni

3.2.2. Chemoenzymatic Synthesis of UDP-4-Ketosugar

3.2.3. Chemoenzymatic Synthesis of UDP-4-Aminosugar

3.2.4. Synthetic Acetylation Toward UDP-diNAcBac

3.2.5. Purification of PglD From C. jejuni and Chemoenzymatic Synthesis of UDP-diNAcBac

4. Chemoenzymatic Preparation of UDP-6-Deoxy-2,4-diNAc-l-Alt With PseB, PseC, and PseH

4.1. Discussion

4.2. Procedure

4.2.1. Enzyme Expression and Purification

4.2.2. Chemoenzymatic Synthesis of UDP-6-deoxy-4-amino-2-NAc-l-Alt

4.2.3. Synthetic Acetylation Toward UDP-6-deoxy-2,4-diNAc-l-Alt

4.2.4. Chemoenzymatic Acetylation Toward UDP-6-deoxy-2,4-diNAc-l-Alt With PseH

5. Chemoenzymatic Preparation of UDP-2,3-diNAcGlcA With WbpB, WbpE, and WbpD

5.1. Discussion

5.2. Procedure

5.2.1. Enzyme Expression and Purification

5.2.2. Synthesis and Purification of UDP-GlcNAcA

5.2.3. Synthesis and Purification of UDP-2-NAc-3-NH2GlcA by the Coupled Reaction of WbpB and WbpE

5.2.4. Acetylation With WbpD Toward UDP-2,3-diNAcGlcA

5.2.5. Analysis of Reaction Products by CE

6. Isolation and Synthesis of Polyprenol-Phosphate Carrier Dol-P

6.1. Discussion

6.2. Procedure

6.2.1. Synthesis of (S)-Dolichols.

6.2.2. Synthesis of (S)-Dolichol Phosphate

6.2.3. Dol-P-GlcNAc-β-(1-3)-2,3-diNAcGlcA Synthesis With AglK and AglC

7. Conclusions

Chapter Six: Biosynthesis of Legionaminic Acid and Its Incorporation Into Glycoconjugates

2. Production of CMP-Legionaminic Acid

3. Sialyltransferases as Tools for the Synthesis of Glycoconjugates With Legionaminic Acid

3.1. Transfer of Legionaminic Acid to Small Synthetic Glycan Acceptors

3.1.1. Procedure for the Synthesis of Leg5,7Ac2-α2-3-Lactosyl Derivatives

3.2. Transfer of Legionaminic Acid to Glycoproteins

3.2.1. Procedure for the Transfer of Leg5,7Ac2 to Asialo-α1-Antitrypsin

3.2.2. Procedure for the Transfer of Leg5,7Ac2 to Asialo-Interferon-α2b

Chapter Seven: Profiling of Sugar Nucleotides

2. Sugar Nucleotide Extraction

3. Sugar Nucleotide Profiling Strategies

3.1. Anion-Exchange Chromatography and UV Detection

3.2. IP-RP HPLC and UV Detection

3.3. Capillary Electrophoresis and UV Detection

3.4. Liquid Chromatography-MS-Based Methods

3.4.1. AEC-MS-Based Methods

3.4.2. IP-RP HPLC-MS-Based Methods

3.4.3. Hydrophilic Interaction Liquid Chromatography-MS-Based Methods

3.4.4. Porous Graphitic Carbon LC-MS-Based Methods

3.5. CE-MS-Based Methods

3.6. High-Throughput Methods

4. Sugar Nucleotide Profiling Protocol

4.1. Cell Culture and Lysis

4.2. Sample Preparation

4.3. ESI-MS/MS on Xevo TQ-S

4.4. Surface-Conditioned PGC LC-MS/MS

4.5. Example of Results

5. Summary

Chapter Eight: Syntheses of Fluorescent Gangliosides for the Studies of Raft Domains

2. Design and Synthetic Strategy

3. Synthesis of Fluorescent Ganglioside Analogs.

3.1. Synthesis of Fluorescent GM3 Analogs.

Notes:

Includes bibliographical references at the end of each chapters and indexes.

Description based on online resource; title from PDF title page (ebrary, viewed October 17, 2017).

ISBN:

9780128114704

0128114703

9780128114698

012811469X