Cell Free Protein Expression 1st Edition by W Antoni Kudlicki ISBN 2007040344 9782007040340

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ISBN 10: 2007040344 
ISBN 13: 9782007040340
Author: W Antoni Kudlicki

Cell Free Protein Expression 1st Table of contents:

Chapter 1 The Role of Cell-Free Rabbit Reticulocyte Expression Systems in Functional Proteomics
Abstract
Abbreviations
Introduction
Membrane Topology
Synthesis of Membrane Proteins
Protease Protection Assays
Tagging Membrane Proteins to Determine Orientation
Glycosylation
N-Linked Glycosylation of Membrane and Secreted Proteins
O-linked Glycosylation of Cytosolic and Nuclear Proteins
Lipid Modification and Acetylation of Proteins
Glycosylphosphatidyl Inositol (GPI) Anchors
Prenylation
N-Myristoylation and Palmitoylation
N-Acetylation
Reconstituting ER-Associated Protein Degradation
In Vitro Viral Assembly
Protein Microarray Technology
Protein Interaction with Other Molecules
Display Technologies
Eukaryotic Ribosome Display
mRNA Display or in Vitro Virus
In Vitro Compartmentalization
Screening
In Vitro Expression Cloning (IVEC)
Protein Truncation Test
Other Screening
Conclusions
References
Chapter 2 Advances in Insect-Based Cell-Free Protein Expression
Abstract
Introduction
Materials and Methods
Construction of DNA Templates
Protein Expression
Protein Analysis
Luciferase Activity Assay
Strep Tag Pull-Down Assay
Protein Deglycosylation
Results and Discussion
Construct Screening
Soluble Expression of Human Proteins
Protein Function
Posttranslationally Modified Proteins
Conclusion
References
Chapter 3 Advantages and Applications of the Batch-Formatted E. coli Cell-Free Expression System
Abstract
Introduction
High-Yield Formats
Membrane Exchange Systems
Longer-Lived Batch Systems
New Configurations
High-Throughput Cell-Free Expression of Fluorescently Labeled Proteins
Automation
Bacterial Protein Arrays
Membrane Protein Expression
NMR Analysis
The Future
Acknowledgements
References
Chapter 4 Energetics in Escherichia coli-Based Batch Cell-Free Systems
Abstract
Introduction
History
Cell Extracts
In Vitro VS in Vivo
Components of E. coli-iixi Cell-Free System
Energy Sources for Cell-Free Protein Expression Systems
Phosphoenolpyruvate
Pyruvate
Glucose-6-Phosphate
3-Phosphoglycerate
Glucose
Other Phosphate Containing Secondary Energy Sources
Secondary Energy Source from Citric Acid Cycle
Large-Scale Protein Production in Batch Reaction
Conclusion
Acknowledgements
References
Chapter 5 Disulfide Bond Formation in Bacteria-Based*Cell-Free Protein Expression
Abstract
Introduction
Mammalian Protein Therapeutics and Vaccines
Comparing Cellular and Cell-Free Production of Disulfide-Bonded Proteins
Expression of Disulfide Bonded Proteins in a Commercially Viable Cell-Free System
Batch Mode Production in a Bacterial Cell-Free System
Amino Acid Stabilization by Genetic Modification of theE. coli Genome
Elimination of Glutathione Reductase in theE. coli Cell-Extract
Using Glucose as the Energy Source for Disulfide Bond Containing Proteins
Scale-Up of Cell-Free Batch Systems
Conclusions
Acknowledgements
References
Chapter 6 The PURE System: A Minimal Cell-Free Translation System
Abstract
Introduction
Construction of the PURE System for Protein Generation
Composition of the PURE System
Productivity of the PURE System
Advantages of the PURE System
Reconstitution of the PURE System for Protein Maturation Studies
Functional Production of Single-Chain Antibodies
Precise Investigation of Protein-Folding Mechanisms
Concluding Remarks
References
Chapter 7 Cell-Free Expression Approaches for the Production and Characterization of Membrane Proteins
Abstract
Introduction
Why Is Cell-Free Expression Exceptionally Suitable for the High-Level Production of Membrane Proteins?
Cell-Free Produced Precipitates: A Fast and Reliable Approach for the Production of High Amounts of Pure Membrane Protein Samples
Cell-Free Expression into Detergent Micelles: A Completely New and Unique Mode of Membrane Protein Production
Cell-Free Expression of Membrane Proteins into Preformed Artificial Liposomes: A Challenging Perspective of Future Developments
Membrane Protein Targets Suitable for Cell-Free Expression
Throughput Approaches and Optimization Strategies for the Production of MPs
Synergies of Cell-Free Expression and the Structural Analysis of Membrane Proteins by NMR Techniques
Conclusions
Acknowledgements
References
Chapter 8 Cell-Free Expression for Protein NMR
Abstract
Introduction
A Bird’s Eye View of NMR Structure Determination
Practical Limits for NMR Structure Determination
Cell-Free Protein Expression
Amino Acid Specific Labeling by Cell-Free Expression
Optimized Labeling Schedules for Backbone Assignment
Other Combinatorial Labeling Schemes
More Exotic Labeling Schemes
Specific Information
Future Prospects
Acknowledgements
References
Chapter 9 Cell-Free Synthesis of Membrane Proteins for X-Ray Crystallography
Abstract
Introduction
Results
Effects of Detergents and Lipids on the Cell-Free Protein Reaction
Increased Solubility of et-HelicalMembrane Proteins
Efficient Labeling of Membrane Proteins with Se-Met
Crystallization of EmrE
Concluding Remarks
Materials and Methods
Plasmids
Preparation of Liposomes
In Vitro Protein Synthesis
Protein Purification
Mass Spectroscopy Analysis
Large Scale Se-Met Labeling and X-Ray Data Collection
References
Chapter 10 Bacterial Cell-Free Expression Systems for High-Throughput Protein Production
Abstract
Introduction to High-Throughput Protein Production
Cell-Free Systems for High-Throughput Protein Expression
Bacterial Cell-Free System for High-Throughput Protein Production
Cell-Free Protein Expression in Our Laboratory
Limitations and Potential Solutions
Conclusion
Reference
Chapter 11 Cell-Free Protein Synthesis for Protein Microarrays
Introduction
Protein Content
Yersinia pestis Protein Arrays
Conclusions
References
Chapter 12 Cell-Free Protein Expression Screening and Protein Immobilization Using Protein Microarrays
Abstract
Introduction
Fluorescence-Based Protein Microarray Expression Screens
Array-Based Site-Specific and Traceless Immobilization of Cell-Free Expressed Proteins
Conclusions
Materials and Methods
Cloning and Bacterial Expression
Template Preparation and Cell-Free Reactions
Expression Screening
pMBP-IN
pTXBl-Ic
pIVEX-MBP-IN
Protein Microarray Spotting and Detection
Functionalization of Glass Substrates
Generation of Protein Microarrays for Protein Immobilization
Acknowledgement
References
Chapter 13 Cell-Free Protein Expression Labeling with Fluorophores
Abstract
Introduction
The Basics of tRNA Mediated Expression Labeling
Misaminoacylated tRNAs
Frameshift Suppression
Fluorophore Labeling at the N-Terminus
Fluorophore Labeling at the C-Terminus
Labeling at Specific Amino Acids
Selected Applications oftRNA Mediated Expression Labeling
Incorporating Fluorescent Reporters
Expression Monitoring/Screening and Cellular Imaging
Detecting Interactions, Conformational Changes and Proteolysis
Detection of Truncation Mutations in Gene Products
Future Perspectives
References
Chapter 14 Cell-Free Synthesis of Defined Protein Conjugates by Site-Directed Cotranslational Labeling
Abstract
Protein Conjugates
Cell-Free Systems as a Tool for Protein Conjugate Production
Incorporation Efficiencies of Different Unnatural Amino Acids
Depletion of RF1 from a Cell-Free System Results in Efficient Incorporation of Biocytin
Applications of Site-Specifically and Stoichiometrically Defined Protein Conjugates
Conclusions
Acknowledgements
References
Chapter 15 C-Terminal Labeling of Proteins Using Fluorescently Conjugated Puromycin Derivatives
Abstract
Introduction
The Principle of C-Terminal Fluorescence Labeling Using Puromycin Derivatives
Advantages of C-Terminal Fluor-Puro Labeling Technology
Successful Fluor-Puro Labeling Applications
Fluor-Puro Conjugates: Labeling Efficiency and Choice of Fluorophore
The Choice of The Translation System
Concentration of Fluor-Puro
Advanced Applications for Fluor-Puro Labeling
FRET System
Increase the Label of Matured Proteins
Selection of Multi Interaction Proteins
Proteomics Analysis
Library Screeningfor Functional Protein
Conclusion
Acknowledgement
References
Chapter 16 Translation Engineering and Synthetic Biology
Abstract
Translation Engineering™—Codon Context and Translational “Pausing”
Codon Pairs Control Translation Kinetics
Why Is Translational Pausing Important?
The CODA Translational Engineering Toolbox
Overview
Mechanisms and Utilities
Tool 1: SpeedPlot™
Tool 2: Hot-rod lM Genes
Tool 3: Pause Conservation Maps™
Tool 4: Planned Pause Gene Sets™
Summary and Applications
References
Chapter 17 Accelerated Protein Evolution Using Ribosome Display
Abstract
Introduction to in Vitro Display Technology
The Ribosome Display Process
Cell-Free Translation for Ribosome Display
In Vitro Evolution by Ribosome Display
Evolution of High Affinity Antibodies
Evolution of Improved Proteins
Conclusion
Acknowledgements
References
Chapter 18 Application of in Vitro Virus (IVV) Technique for High-Throughput Analysis of Protein-Protein Interactions
Abstract
Introduction
IVY: Genotype-Phenotype Assignment Molecules Formed via Puromycin
Applications of IVV as in Vitro Display Technology
Protein Complexes Analysis; a Co-Translation Technique
Reliability of IVV Data; the Problem of False Positives and Negatives
In Silico Analysis ofIVV Selection for AP-1 (Fos/Jun) Complex

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W Antoni Kudlicki,Protein Expression