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Chemical glycobiology. Part A, Synthesis, manipulation and applications of glycans /
Awareness of the biological significance of glycans and glycoconjugates continues to grow by leaps and bounds. Taking a cue from chemical biology, in general, the volumes in this two part treatment present strategies and methods from diverse fields of biochemistry, biophysics, bioinformatics, organi...
| Clasificación: | Libro Electrónico |
|---|---|
| Otros Autores: | |
| Formato: | Electrónico eBook |
| Idioma: | Inglés |
| Publicado: |
Cambridge :
Academic Press,
[2017]
|
| Colección: | Methods in enzymology ;
v. 597. |
| Temas: | |
| Acceso en línea: | Texto completo |
Tabla de Contenidos:
- 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 Reagents3.1.3. 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; References
- Chapter Two: Structure-Guided Directed Evolution of Glycosidases: A Case Study in Engineering a Blood Group Antigen-Cleav ... 1. Introduction; 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); 2.2.1. Equipment; 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. Notes3. Enzymatic Preparation of Fluorogenic Oligosaccharides as Substrates for High-Throughput Assays; 3.1. In Vitro Enzymatic Synthesis of Fluorogenic Oligosaccharides; 3.1.1. Equipment; 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 Glycosidases4.2.1. Equipment and Software Tools; 4.2.2. Buffers, Solutions, and Reagents; 4.2.3. Procedure; 4.2.4. Notes; 5. Summary and Conclusion; Acknowledgments; References; Chapter Three: A Pipeline for Studying and Engineering Single-Subunit Oligosaccharyltransferases; 1. Introduction; 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