Isotope Labeling of Biomolecules - Labeling Methods

 
 
Academic Press
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  • erschienen am 26. November 2015
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Isotope Labeling of Biomolecules - Labeling Methods, the latest volume of the Methods in Enzymology series contains comprehensive information on stable isotope labeling methods and applications for biomolecules.
  • Contains contributions from leading authorities in the field of isotope labeling of biomolecules
  • Informs and updates on the latest developments in the field
  • Provides comprehensive information on stable isotope labeling methods and applications for biomolecules
0076-6879
  • Englisch
  • USA
Elsevier Science
  • 22,51 MB
978-0-12-803080-6 (9780128030806)
0128030801 (0128030801)
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  • Front Cover
  • Isotope Labeling of Biomolecules - Labeling Methods
  • Copyright
  • Contents
  • Contributors
  • Preface
  • Section I: Labeling in Prokarya
  • Chapter One: Robust High-Yield Methodologies for 2H and 2H/15N/13C Labeling of Proteins for Structural Investigations Usi...
  • 1. Introduction
  • 2. Media Preparation
  • 3. Unlabeled Protein Production
  • 3.1. Transformation of Expression Cells and Staged Culturing
  • 3.1.1. Transformation
  • 3.1.2. Flask Culture 1
  • 3.1.3. Flask Culture 2
  • 3.1.4. Flask Culture 3
  • 3.2. 1L Bioreactor Culture
  • 3.2.1. Bioreactor Set-Up and Inoculation
  • 3.2.2. Running the Bioreactor to Induction and Harvest
  • 3.2.3. Growth Rate Expectations and Prediction
  • 3.2.4. SDS-PAGE Analysis of Expression Control and Expression Level
  • 4. Deuterated Protein Production
  • 4.1. Deuteration in 90% D2O for SANS
  • 4.1.1. Culture Adaptation to 50% D2O then 90% D2O
  • 4.2. Deuteration in 100% D2O with Unlabeled Glycerol
  • 4.2.1. Culture Adaptation to 90% D2O then 100% D2O
  • 4.3. Perdeuteration: 100% D2O and Deuterated Carbon Source
  • 4.4. Deuteration Level Quantification
  • 5. Multiple Labeling of Proteins for NMR
  • 5.1. 15N Labeling
  • 5.2. 13C Labeling
  • 5.2.1. Perdeuteration with 13C Labeling
  • 6. Comments on the Method
  • 6.1. Plasmid Choice
  • 6.2. Precipitating Media
  • 6.3. Use of Commercial Supercompetent Expression Cells
  • 6.4. Staged Starter Cultures
  • 6.5. Gentle Handling of the Cultures
  • 6.6. 15N Ammonium Hydroxide or Sodium Hydroxide as Base Feed for pH Control
  • 7. Typical Deuteration Levels
  • Acknowledgments
  • References
  • Chapter Two: Protein Labeling in Escherichia coli with 2H, 13C, and 15N
  • 1. Introduction
  • 2. Selection of Induction Method
  • 2.1. Manual Induction
  • 2.2. Autoinduction
  • 2.3. Manual Versus Autoinduction
  • 3. Special Considerations When Labeling with Deuterium
  • 4. Plasmid and E. coli Strain Selection
  • 5. Determination of Isotope Incorporation
  • 6. Acclimation of E. coli to Growth in D2O
  • 7. Media Preparation
  • 7.1. Stock Solutions
  • 7.1.1. 20x Modified Neidhardt Stock
  • 7.1.2. 20x Nitrogen Phosphate Stock
  • 7.1.3. 50x Carbon Source Stock
  • 7.1.4. 50x Autoinduction Carbon Source Stock
  • 7.1.5. 10,000x Trace Elements Stock
  • 7.1.6. 1000x Vitamin Stock
  • 7.2. Media Composition
  • 7.2.1. Modified Neidhardt Media
  • 7.2.2. Modified Tyler Media
  • 7.2.3. Modified Tyler Medium for Autoinduction
  • 8. Protein Expression
  • 8.1. Manual Induction
  • 8.1.1. Day 1
  • 8.1.2. Day 2
  • 8.2. Autoinduction
  • 8.2.1. Day 1
  • 8.2.2. Day 2
  • 8.2.3. Day 3
  • Acknowledgments
  • References
  • Chapter Three: Escherichia coli Auxotroph Host Strains for Amino Acid-Selective Isotope Labeling of Recombinant Proteins
  • 1. Introduction
  • 2. E. coli Auxotrophs for Amino Acid-Selective Isotope Labeling
  • 2.1. Selection of Appropriate E. coli Auxotroph Strains
  • 2.2. Challenges for Amino Acid-Selective Isotope Labeling
  • 3. Methods
  • 3.1. Homologous Expression and Amino Acid-Selective Labeling of a Membrane Protein Complex Cyt bo3 with E. coli Auxotrophs
  • 3.2. Heterologous Expression and Preparation of the Selectively 15Ne-Glutamine-Labeled FdxB of P. putida JCM 20004 (On th ...
  • 3.3. Heterologous Expression and Preparation of 14N(In Natural Abundance, N/A) Lysine-Labeled ARF on the 15N-Protein Back ...
  • 3.4. Heterologous Expression and Preparation of 14N(N/A) Tyrosine-Labeled ARF on the 15N-Protein Background by Reverse La ...
  • 4. Conclusions
  • Acknowledgments
  • References
  • Chapter Four: 19F-Modified Proteins and 19F-Containing Ligands as Tools in Solution NMR Studies of Protein Interactions
  • 1. Introduction
  • 2. Protocol 1: Biosynthetic Amino Acid Type-Specific Incorporation of 19F-Modified Aromatic Amino Acids
  • 2.1. Protocol Overview
  • 2.2. Step 1: Preparation of Constructs and Transformation
  • 2.3. Tip
  • 2.4. Step 2: Expression of m-Fluoro-l-Tyrosine-Containing Protein
  • 2.5. Tip
  • 2.6. Tip
  • 2.7. Tip
  • 3. Protocol 2: Site-Specific Incorporation of Fluorinated Amino Acids Using a Recombinantly Expressed Orthogonal Amber tR...
  • 3.1. Protocol Overview
  • 3.2. Step 1: Preparation of Constructs and Transformation
  • 3.3. Tip
  • 3.4. Tip
  • 3.5. Tip
  • 3.6. Step 2: Expression of 4-(Trifluoromethyl)-Phenylalanine-Containing Protein
  • 3.7. Tip
  • 3.8. Tip
  • 4. General Considerations for 19F-Observe NMR Experiments
  • 4.1. Solubility and Stability of the NMR Sample
  • 4.2. Spectrometer Magnetic Field Strength
  • 5. 19F-Modified Protein-Observe NMR Experiments
  • 5.1. Protein-Ligand Interactions
  • 5.2. Protein Un/Folding
  • 5.3. Protein Aggregation
  • 5.4. Protein-Lipid Interactions
  • 6. NMR Experiments with 19F-Containing Ligands
  • 6.1. Monitoring Line Broadening
  • 6.2. Competition-Based Experiments
  • 6.3. 19F NMR-Based Biochemical Screening
  • 6.4. Magnetization Transfer Experiments
  • Acknowledgments
  • References
  • Chapter Five: Biopolymer Deuteration for Neutron Scattering and Other Isotope-Sensitive Techniques
  • 1. Introduction
  • 2. Deuterated Biopolyesters
  • 2.1. Poly-3-Hydroxybutyrate Properties and Characterization
  • 2.2. Production of Deuterated PHB
  • 2.3. Polymer Extraction and Analysis
  • 2.4. Poly-3-Hydroxyoctanoate Biodeuteration for Characterization Studies
  • 2.5. PHO Production and Deuteration
  • 2.6. Selective Deuteration to Probe Metabolic Pathways
  • 3. Deuterated Chitosan
  • 3.1. Chitosan from Pichia pastoris
  • 3.2. Deuteration of P. pastoris and Chitosan
  • 3.3. Biomass Production and Deuterium Adaptation
  • 3.3.1. Media Recipes
  • 3.3.1.1. YPD Medium
  • 3.3.1.2. Buffered Minimal Methanol Medium
  • 3.3.2. High Cell Density Growth of P. pastoris on Methanol
  • 3.3.3. D2O Adaptation and Production of Deuterated Biomass
  • 3.4. Chitosan Extraction and Purification
  • 3.5. Characterization of Chitosan Biodeuteration
  • 3.6. Final Remarks
  • 4. Deuterated Cellulose
  • 4.1. Organization and Biosynthesis of Bacterial Cellulose
  • 4.2. Production and Characterization of BC
  • 4.3. Final Remarks
  • Acknowledgments
  • References
  • Chapter Six: Production of Bacterial Cellulose with Controlled Deuterium-Hydrogen Substitution for Neutron Scattering Stu ...
  • 1. Introduction
  • 1.1. Neutrons in Biology
  • 1.2. Biological Toxicity of Deuterium Oxide
  • 2. The Occurrence and Properties of Cellulose
  • 3. Deuteration of Bacterial Cellulose
  • 3.1. Choice of Bacterial Strain
  • 3.2. Growth Media for Deuterated Cellulose Production
  • 3.3. Adaptation of Cell Growth in Deuterium Oxide
  • 3.4. Cellulose Purification
  • 4. Characterization of Deuterated Cellulose
  • 4.1. Chemical and Physical Characterization of Bacterial Cellulose
  • 4.2. Fourier Transform Infrared Spectroscopy
  • 4.3. Mass Spectrometry
  • 4.4. SANS Analysis of Bacterial Cellulose
  • Acknowledgments
  • References
  • Chapter Seven: Isotopic Labeling of Proteins in Halobacterium salinarum
  • 1. Introduction
  • 2. Growth and Maintenance of Halobacterium salinarum
  • 2.1. Overview
  • 2.2. Reagents
  • 2.3. Solutions and Buffers
  • 2.4. Starter Culture Growth in Nonlabeled Medium
  • 2.5. Frozen Storage of Cultures
  • 2.6. Growth on Agar Plates
  • 2.7. Growth of Halobacterium salinarum in Isotopically Labeled Medium
  • 3. Purification of Proteins from Halobacterium salinarum
  • 3.1. Factors Affecting Protein Expression
  • 3.2. Cell Growth for Protein Production
  • 3.3. Cell Harvest
  • 3.4. Cell Lysis
  • 3.4.1. General Considerations
  • 3.4.2. Low-Salt Lysis
  • 3.4.3. French Press, Microfluidizer, or Similar
  • 3.4.4. Sonication Lysis
  • 3.5. Expression and Purification of Bacteriorhodopsin
  • 3.5.1. Solutions
  • 3.5.2. Expression
  • 3.5.3. Lysis and Membrane Harvest
  • 3.5.4. Sucrose Gradient Ultracentrifugation
  • 3.5.5. Detergent Solubilization and Gel Filtration
  • 3.6. Verifying Isotopic Labeling of Bacteriorhodopsin by MALDI-MS
  • 3.6.1. Protein Calibration Standards
  • 4. Summary
  • Acknowledgments
  • References
  • Chapter Eight: Amino Acid Selective Unlabeling in Protein NMR Spectroscopy
  • 1. Introduction
  • 2. Method Description
  • 2.1. Sample Preparation
  • 2.2. Identification of Peaks
  • 2.3. Isotope Scrambling
  • 2.4. Choice of Amino Acid Types for Selective Unlabeling
  • 2.4.1. Optimal Combination of Amino Acid Types for Selective Unlabeling
  • 2.5. A Method for Sequential Assignments Using Selectively Unlabeling
  • 3. Applications of Selective Unlabeling
  • 4. Conclusions
  • Acknowledgments
  • References
  • Section II: Labeling in Eukarya
  • Chapter Nine: Isotope Labeling of Eukaryotic Membrane Proteins in Yeast for Solid-State NMR
  • 1. Introduction
  • 2. Expression and Isotope Labeling in P. pastoris: Background
  • 3. Isotope Labeling of Membrane Proteins in P. pastoris
  • 3.1. Protein Targets and Vectors
  • 3.2. Optimization of Small-Scale Natural Abundance Expression
  • 3.3. Large-Scale Isotope Labeling
  • 3.4. Production of Samples for ssNMR
  • 4. Outlook
  • Acknowledgments
  • References
  • Chapter Ten: Development of Approaches for Deuterium Incorporation in Plants
  • 1. Introduction
  • 1.1. Applications of Deuterium Labeling in Plants
  • 2. Challenges of Plant Cultivation in D2O
  • 2.1. Inhibitory Effects of D2O on Plants
  • 2.2. Chemical and Physical Properties of D2O
  • 2.3. Differences Between Plant Growth in D2O and H2O
  • 2.4. Natural Variation of D2O Tolerance in Plant Species
  • 3. Analysis of Deuterium-Labeled Plant Biomass
  • 3.1. Analysis of Deuterium Substitution
  • 3.2. Chemical and Physical Characterization of Lignocellulosic Biomass
  • 4. Deuterium Labeling of Plants for Metabolic Studies
  • 4.1. Deuteration Studies of Duckweed
  • 4.2. Isotopic Labeling of Plants for Nutritional Tracer Studies
  • 4.3. Development of Arabidopsis as a Platform for Deuterium Tracing
  • 5. Production of Deuterated Plants for Structural Studies
  • 5.1. Methods for Cultivation of Plants in High Concentrations of D2O
  • 5.2. Multiple-Chamber Perfusion System for Long-Term Cultivation
  • 5.3. Production of Deuterated Annual Ryegrass
  • 5.4. Production of Deuterated Switchgrass
  • 5.5. Deuterium Labeling Winter Grain Rye with D2O and Deuterated Phenylalanine
  • Acknowledgments
  • References
  • Chapter Eleven: Isotope Labeling of Proteins in Insect Cells
  • 1. Insect Cells as Expression System
  • 1.1. Comparison to Other Expression Systems
  • 1.2. Insect Cell Cultures and Their Use as Expression System
  • 1.3. The Baculovirus as a Vehicle for Efficient Transfection of Insect Cells
  • 1.3.1. The Baculovirus Life Cycle
  • 1.3.2. Virus Generation Protocols
  • 1.4. Optimization of Yields
  • 2. General Considerations for Isotope Labeling in Insect Cells
  • 2.1. Sources of Amino Acids in Insect Cell Media
  • 2.2. Factors Influencing Isotope Incorporation Levels
  • 2.2.1. Carry Over from Preculture
  • 2.2.2. Unlabeled Medium from Baculovirus Suspension
  • 2.2.3. Adverse Effects from Insect Cell Metabolism
  • 3. Amino Acid Type-Specific Isotope Labeling in Insect Cells
  • 3.1. Scrambling Versus Label Dilution
  • 3.2. Experimental Approaches
  • 3.2.1. Replacing Amino Acids
  • 3.2.2. Excess Labeling
  • 3.2.3. Chemical Labeling of Expressed Proteins
  • 4. Uniform Isotope Labeling in Insect Cells
  • 4.1. Experimental Approaches
  • 4.1.1. Commercial Media
  • 4.1.2. Economic Media Based on Amino Acid Extracts
  • 4.2. Uniform 15N Labeling
  • 4.3. Uniform 13C Labeling
  • 4.4. Uniform 2H Labeling
  • 4.4.1. Approaches
  • 4.4.2. Relevant Metabolism for 2H Labeling
  • 5. Applications
  • 5.1. Uniform Labeling for Structural Studies
  • 5.2. Membrane Proteins
  • 5.3. In-Cell NMR
  • 5.4. Applications in Drug Discovery
  • 6. Protocols
  • 6.1. Materials
  • 6.1.1. Insect Cell Lines
  • 6.1.2. Media
  • 6.1.3. Reagents
  • 6.1.4. Consumables
  • 6.1.5. Equipment
  • 6.2. Culturing of Insect Cells
  • 6.2.1. Starting an Insect Cell Culture
  • 6.2.2. Generation of Stock Cell Lines
  • 6.3. Transfection and Infection of Insect Cells with Baculovirus
  • 6.3.1. Virus Amplification
  • 6.4. General Protocol for Amino Acid-Type Selective Isotope Labeling in Insect Cells
  • 6.4.1. Recipe for 1L of Medium for Amino Acid-Type Selective Labeling
  • 6.4.2. Protocol
  • 6.5. General Protocol for Uniform Isotope Labeling in Insect Cells
  • 6.5.1. Recipe for 1L of Medium for Uniform Isotope Labeling in Insect Cells
  • 6.5.2. Protocol
  • References
  • Chapter Twelve: Effective Isotope Labeling of Proteins in a Mammalian Expression System
  • 1. Introduction
  • 2. Overview of Mammalian Expression
  • 2.1. Transient Protein Expression in Mammalian Cells
  • 2.1.1. Procedure for Transient Expression of Proteins and Protein Domains in Mammalian Cells
  • 2.1.2. Protein Expression and Construct Selection
  • 2.2. Transient Protein Expression Using Mammalian Viruses
  • 2.2.1. Construction of the Adenoviral Shuttle Vector
  • 2.2.2. Generation of Recombinant Adenoviral Genome Containing the GOI
  • 2.2.3. Generation of Adenoviruses Containing GOI
  • 3. Protein Expression
  • 3.1. Selective Labeling of Specific Amino Acids
  • 4. NMR Characterization of Expressed Protein
  • 5. Conclusions
  • 6. Materials
  • Acknowledgments
  • References
  • Section III: In Vitro Labeling
  • Chapter Thirteen: Escherichia coli Cell-Free Protein Synthesis and Isotope Labeling of Mammalian Proteins
  • 1. Introduction
  • 2. The E. coli Cell-Free Protein Synthesis Method
  • 2.1. Coupled Transcription-Translation
  • 2.2. Reaction Modes
  • 2.3. Coding Region
  • 2.4. Tags
  • 2.5. Template DNA
  • 2.6. T7 RNA Polymerase
  • 2.7. S30 Extract and tRNAs
  • 2.8. Low-Molecular Mass Components
  • 2.9. Disulfide Bonds
  • 2.10. Molecular Chaperones
  • 2.11. Ligand Complexes
  • 3. Stable Isotope Labeling of Proteins
  • 3.1. Stable Isotope-Labeled Amino Acids
  • 3.2. Inhibitors of Amino Acid Metabolism
  • 3.3. Site-Directed Labeling
  • 3.4. Constructs and Cell-Free Reaction Conditions
  • 3.5. Medium-Scale Production of 15N-Labeled Proteins
  • 3.6. Large-Scale Production of 13C/15N-Labeled Proteins
  • 3.7. Purification of Synthesized Proteins
  • 3.8. WWE Domains
  • 3.9. Zinc-Binding Proteins
  • 3.10. Peptide Complexes
  • Acknowledgments
  • References
  • Chapter Fourteen: Rapid Biosynthesis of Stable Isotope-Labeled Peptides from a Reconstituted In Vitro Translation System ...
  • 1. Introduction
  • 2. Equipment, Materials, and Buffers
  • 3. Section1: DNA Template Preparation
  • 3.1. Overview
  • 3.2. PCR Amplification of Double-Strand DNA Template for Peptide Expression
  • 3.3. Tips
  • 4. Section2: Peptide Synthesis with PURE System
  • 4.1. Overview
  • 4.2. Translation Using PURE System
  • 4.3. Tips
  • 5. Section3: Enrichment and Digestion of Synthesized Peptide
  • 5.1. Overview
  • 5.2. Purification and Digestion of PURE-Synthesized Peptide
  • 5.3. Tips
  • 6. Section4: Quantification of PURE-Synthesized Peptide
  • 6.1. Overview
  • 6.2. Quantification of PURE-Expressed Peptides for Absolute Quantification
  • 6.3. Tips
  • 7. An Example
  • 8. Summary and Discussion
  • Acknowledgment
  • References
  • Chapter Fifteen: Labeling of Membrane Proteins by Cell-Free Expression
  • 1. Introduction
  • 2. Core Considerations for the Cell-Free Generation of MP Samples
  • 3. Specific Challenges of NMR Studies with MPs
  • 4. An Emerging Perspective: NMR with NDs
  • 5. Labeling of Cell-Free Synthesized MPs with Stable Isotopes
  • 6. Reducing Scrambling Problems
  • 7. Perdeuteration of Cell-Free Synthesized MPs
  • 8. Conclusion
  • Acknowledgments
  • References
  • Chapter Sixteen: Selective Amino Acid Segmental Labeling of Multi-Domain Proteins
  • 1. Introduction
  • 1.1. Overview
  • 1.2. Segmental Isotope Labeling
  • 1.2.1. Native Chemical Ligation
  • 1.2.2. Expressed Protein Ligation
  • 1.2.3. Protein trans-Splicing
  • 1.2.4. Sortase-Mediated Protein Ligation
  • 1.3. Amino Acid-Specific Labeling
  • 1.4. Segmental Amino Acid-Type Isotope Labeling
  • 2. Methods
  • 2.1. Methodological Approach
  • 2.2. Important Requirements for Expressed Protein Ligation
  • 2.3. Choosing an Optimal Ligation Site
  • 2.3.1. Importance of the Ligation Site
  • 2.3.2. Guidelines to an Optimal Ligation Site
  • 2.4. Production of Precursor Constructs
  • 2.4.1. Overview
  • 2.4.2. Cloning of Target Protein Fragments
  • 2.4.3. Precursor Production in Escherichia coli
  • 2.4.4. Precursor Production with Cell-Free Expression
  • 2.5. Ligation of Precursor Fragments
  • 2.5.1. Overview
  • 2.5.2. Ligation of Purified Precursors
  • 2.5.3. On-Column Ligation
  • 2.6. Practical Example: Amino Acid-Selective Segmental Labeling of a Multi-Domain RNA-Binding Protein
  • 3. Conclusion
  • Acknowledgments
  • References
  • Chapter Seventeen: Labeling Monosaccharides With Stable Isotopes
  • 1. Introduction
  • 2. Terminology to Describe Different Monosaccharide Isotopomers
  • 3. Introducing 13C into Monosaccharides
  • 3.1. Cyanohydrin Reduction
  • 3.2. Permutations of Cyanohydrin Reduction
  • 3.2.1. Generalized Laboratory Protocol-Cyanohydrin Reduction Reaction
  • 3.3. Molybdate-Catalyzed Epimerization of Aldoses Accompanied by C1-C2 Transposition
  • 3.3.1. Generalized Laboratory Protocol-Molybdate-Catalyzed Epimerization of Aldoses
  • 4. Multiple Labeling of Aldoses Via Chain Inversion
  • 5. Labeling at the Internal Carbons of Aldoses
  • 6. Extension to Biologically Important Aldoses
  • 7. Relative Carbonyl Reactivities in Osones-Synthesis of Labeled 2-Ketoses
  • 8. Manipulation of Three-Carbon Building Blocks in Enzyme-Mediated Aldol Condensation
  • 9. Manipulation of Isotopically Labeled d-Fructose 17 and l-Sorbose 25
  • 10. Concluding Remarks
  • References
  • Section IV: RNA Labeling
  • Chapter Eighteen: Stable Isotope-Labeled RNA Phosphoramidites to Facilitate Dynamics by NMR
  • 1. Theory
  • 2. Equipment
  • 3. Materials
  • 3.1. Solutions and Buffers
  • 4. Protocol
  • 4.1. Duration
  • 4.2. Preparation
  • 4.3. Caution
  • 5. Step 1: Synthesis of 6-13C-Uridine TOM Phosphoramidite
  • 5.1. Overview
  • 5.2. Duration
  • 5.2.1. Tip
  • 5.2.2. Tip
  • 5.2.3. Tip
  • 5.2.4. Tip
  • 5.2.5. Tip
  • 5.2.6. Tip
  • 5.2.7. Tip
  • 6. Step 2: Synthesis of 6-13C-Cytidine TOM Phosphoramidite
  • 6.1. Overview
  • 6.2. Duration
  • 6.2.1. Tip
  • 6.2.2. Tip
  • 6.2.3. Tip
  • 7. Step 3: Chemical RNA Synthesis
  • 7.1. Overview
  • 7.2. Duration
  • 7.2.1. Tip
  • 7.2.2. Tip
  • 7.2.3. Tip
  • 7.2.4. Tip
  • 8. Step 4: Applications
  • 8.1. Overview
  • 8.2. Segmental Stable Isotope Labeling Using Chemically Synthesized RNAs
  • 8.3. 13C CPMG RD NMR Spectroscopy
  • 8.4. 13C ZZ Exchange NMR Spectroscopy
  • 8.5. Chemical Exchange Saturation Transfer
  • 9. Conclusions
  • Acknowledgments
  • References
  • Chapter Nineteen: In Vivo, Large-Scale Preparation of Uniformly 15N- and Site-Specifically 13C-Labeled Homogeneous, Recom ...
  • 1. Theory
  • 2. Equipment
  • 3. Materials
  • 3.1. Solutions for Bacterial Growth
  • 3.2. Solutions for Recombinant tRNA-Scaffold Purification
  • 3.3. Solution for DNAzyme Cleavage
  • 3.4. Solutions for Denaturing PAGE
  • 4. Protocol
  • 4.1. Duration
  • 4.2. Preparation
  • 4.3. Caution
  • 5. Step 1: Pilot of the Expression of the Recombinant tRNA-Scaffold Plasmid in Wild-Type K12 E. coli
  • 5.1. Overview
  • 5.2. Duration
  • 5.3. Caution
  • 5.4. Tip
  • 5.5. Tip
  • 5.6. Tip
  • 6. Step 2: Double Selection of High-Expressing E. coli Clones
  • 6.1. Overview
  • 6.2. Duration
  • 6.3. Tip
  • 6.4. Tip
  • 6.5. Tip
  • 6.6. Tip
  • 6.7. Tip
  • 6.8. Tip
  • 7. Step 3: Large-Scale Expression in Labeled SPG Minimal Media
  • 7.1. Overview
  • 7.2. Duration
  • 7.3. Tip
  • 7.4. Tip
  • 7.5. Tip
  • 7.6. Tip
  • 7.7. Tip
  • 8. Step 4: Total Cellular RNA Extraction
  • 8.1. Overview
  • 8.2. Duration
  • 8.3. Tip
  • 8.4. Tip
  • 8.5. Tip
  • 8.6. Tip
  • 8.7. Tip
  • 8.8. Tip
  • 9. Step 5a: Purification of the Recombinant tRNA-Scaffold Using Anion-Exchange Chromatography
  • 9.1. Overview
  • 9.2. Duration
  • 9.3. Tip
  • 9.4. Tip
  • 9.5. Tip
  • 9.6. Tip
  • 9.7. Tip
  • 9.8. Tip
  • 9.9. Tip
  • 10. Step 5b: Purification of the Recombinant tRNA-Scaffold Using Affinity Chromatography
  • 10.1. Overview
  • 10.2. Duration
  • 11. Step 6: Excision and Purification of the RNA of Interest
  • 11.1. Overview
  • 11.2. Duration
  • 11.3. Tip
  • 11.4. Tip
  • 11.5. Tip
  • 11.6. Tip
  • 11.7. Tip
  • 11.8. Tip
  • 11.9. Tip
  • 12. Step 7: NMR Applications
  • 12.1. Overview
  • 13. Conclusion
  • Acknowledgments
  • References
  • Chapter Twenty: Cut and Paste RNA for Nuclear Magnetic Resonance, Paramagnetic Resonance Enhancement, and Electron Parama...
  • 1. Introduction
  • 2. Cut and Paste RNA Approach
  • 2.1. Principle
  • 2.2. Building Blocks
  • 2.2.1. Spin-Labeled RNA Fragments
  • 2.2.2. Unlabeled and Isotopically Labeled RNA Fragments
  • 2.3. Combination and Ligation
  • 2.3.1. Segmentally Labeled RNAs for NMR Structural Studies
  • 2.3.2. Spin-Labeled RNAs for Pulsed EPR
  • 2.3.3. Isotopically and Spin-Labeled RNAs for PRE NMR
  • 3. Production of Small (<_10nts isotopically="isotopically" labeled="labeled" li="li" rnas="rnas">
  • 3.1. Introduction
  • 3.2. Double RNase H Cleavage
  • 3.3. Combined RNase H and VS Ribozyme Cleavage
  • 4. Protocol A: Production of Small Spin-Labeled RNA Fragments
  • 5. Protocol B: Production of Unlabeled and Isotopically Labeled RNA Fragments
  • 5.1. In Vitro Transcription and Ribozyme Cleavage
  • 5.2. Purification by Denaturing Anion-Exchange HPLC
  • 5.3. Sequence-Specific RNase H Cleavage
  • 5.4. VS Ribozyme Cleavage of Purified RNA Fragments
  • 5.5. Tips
  • 6. Protocol C: Ligation
  • 6.1. Protocol
  • 6.2. Tips
  • 7. Summary and Outlook
  • Acknowledgments
  • References
  • Author Index
  • Subject Index
  • Back Cover

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