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Recombinant Protein Expression: Prokaryotic hosts and cell-free systems

1st Edition - October 28, 2021

Editors: William B. O'Dell, Zvi Kelman

Hardback ISBN:

9 7 8 - 0 - 3 2 3 - 9 0 1 4 6 - 8

eBook ISBN:

9 7 8 - 0 - 3 2 3 - 9 0 1 4 7 - 5

Recombinant Protein Expression, Part A, Volume 659 in the Methods in Enzymology series, highlights new advances in the field with this new volume presenting interesting chapters… Read more

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Recombinant Protein Expression, Part A, Volume 659 in the Methods in Enzymology series, highlights new advances in the field with this new volume presenting interesting chapters on Multiplexed analysis protein: Protein interactions of polypeptides translated in Leishmania cell-free system, MultiBac system and its applications, performance and recent, Production of antibodies in Shuffle, Designing hybrid-promoter architectures by engineering cis-acting DNA sites to enhance transcription in yeast, Designing hybrid-promoter architectures by engineering cis-acting DNA sites to deregulate transcription in yeast, Antibody or protein-based vaccine production in plants, Cell-free protein synthesis, Plant-based expression of biologic drugs, and much more.

Additional sections cover the Use of native mass spectrometry to guide detergent-based rescue of non-native oligomerization by recombinant proteins, Advancing overexpression and purification of recombinant proteins by pilot optimization through tandem affinity-buffer exchange chromatography online with native mass spectrometry, Method for High-Efficiency Fed-batch cultures of recombinant Escherichia coli, Method to transfer Chinese hamster ovary (CHO) shake flask experiments to the ambr® 250, and Expression of recombinant antibodies in Leishmania tarentolae.

Cover image
Title page
Table of Contents
Copyright
Contributors
Preface
Bacterial hosts
Archaeal hosts
Cell-free systems
Section I: Bacterial hosts
Chapter One: Starting a new recombinant protein production project in Escherichia coli
Abstract
1: Introduction
2: Choosing an appropriate E. coli strain
3: E. coli cultivation for recombinant protein production
4: Methods
5: Afterword
Acknowledgments
Chapter Two: From the notebook to recombinant protein production in Escherichia coli: Design of expression vectors and gene cloning
Abstract
1: Introduction
2: Tweaking the coding sequence of interest
3: Selecting a suitable expression vector
4: Working example: Design, construction, and cloning of a CDS
5: Methods
6: Afterword
Acknowledgments
Chapter Three: Use of tandem affinity–buffer exchange chromatography online with native mass spectrometry for optimizing overexpression and purification of recombinant proteins
Abstract
1: Introduction
2: Materials
3: Methods
4: Notes
5: Discussion
6: Summary
Chapter Four: Purification, reconstitution, and mass analysis of archaeal RNase P, a multisubunit ribonucleoprotein enzyme
Abstract
1: Introduction
2: Materials
3: Methods
4: Notes
5: Conclusions
Acknowledgments
Chapter Five: Production of antibodies in SHuffle Escherichia coli strains
Abstract
1: Introduction
2: Equipment and materials
3: Protocol
4: Concluding remarks
5: Notes
Chapter Six: Improved folding of recombinant protein via co-expression of exogenous chaperones
Abstract
1: Introduction
2: Methods
3: Protocol for in vitro deconstruction assay
4: Key resources
5: Materials and equipment
6: Step-by-step method details
7: Morphological characterization of deconstruction assay products
8: Structural characterization of deconstruction assay products
9: Results
10: Discussion
11: Future development
Acknowledgments
Chapter Seven: Fusing an insoluble protein to GroEL apical domain enhances soluble expression in Escherichia coli
Abstract
1: Introduction
2: Before you begin
3: Key resources table
4: Materials and equipment
5: Step-by-step method details
6: Expected outcomes
7: Advantages
8: Limitations
Chapter Eight: Method for high-efficiency fed-batch cultures of recombinant Escherichia coli
Abstract
1: Introduction
2: Before you begin
3: Materials and equipment
4: Step-by-step method details
5: Expected outcomes
6: Advantages
7: Limitations
8: Safety considerations and standards
Acknowledgments
Chapter Nine: Fed-batch production of deuterated protein in Escherichia coli for neutron scattering experimentation
Abstract
1: Introduction
2: Materials and equipment
3: Media preparation and cell adaption
4: Fed-batch production of deuterated protein
5: Expected outcomes
6: Advantages
7: Limitations
8: Safety considerations and standards
9: Alternative methods/procedures
Section II: Archaeal hosts
Chapter Ten: Thermococcus kodakarensis provides a versatile hyperthermophilic archaeal platform for protein expression
Abstract
1: Introduction
2: Genetic systems for Thermococcus kodakarensis
3: Purifying proteins and identifying protein interactions
4: Reagents, recipes and equipment
5: Protocols
6: Advantages and future perspectives
7: Notes
Chapter Eleven: Recombinant protein expression in Sulfolobus islandicus
Abstract
1: Introduction
2: Equipment
3: Materials
4: Step-by-step protocol
5: Expected outcomes
6: Advantages
7: Limitations
8: Optimization and troubleshooting
Acknowledgments
Chapter Twelve: High-level synthesis and secretion of laccase, a metalloenzyme biocatalyst, by the halophilic archaeon Haloferax volcanii
Abstract
1: Introduction
2: Haloarchaea as a resource for biocatalysts
3: Haloarchaea as platform for high-level protein secretion
4: General strategy for Haloferax volcanii to secrete laccase
5: Methods
6: Protocol for laccase production using H. volcanii as the microbial platform
7: Future perspectives
Chapter Thirteen: Expression and tandem affinity purification of 20S proteasomes and other multisubunit complexes in Haloferax volcanii
Abstract
1: Introduction
2: Proteasomes as multisubunit complexes
3: General strategy for use of Haloferax volcanii to express and purify multisubunit complexes
4: Protocols for heterologous expression and purification of multisubunit complexes
5: Future perspectives
Acknowledgments
Chapter Fourteen: Purification and characterization of ribonucleoprotein effector complexes of Sulfolobus islandicus CRISPR-Cas systems
Abstract
1: Introduction
2: Before you begin
3: Key resources table
4: Materials and equipment
5: Consumable items
6: Step-by-step method details
7: Expected outcomes
8: Advantages
9: Limitations
10: Optimization and troubleshooting
11: Safety considerations and standards
Acknowledgments
Section III: Cell-free systems
Chapter Fifteen: Guidelines for nucleic acid template design for optimal cell-free protein synthesis using an Escherichia coli reconstituted system or a lysate-based system
Abstract
1: Introduction
2: Requirements for protein synthesis from nucleic acid templates
3: Types of templates
4: Protocols
5: Results
6: Summary
Acknowledgment
Chapter Sixteen: Cell-free protein synthesis of CRISPR ribonucleoproteins (RNP)
Abstract
1: Introduction
2: DNA template design for Cas effector and guide RNA
3: Materials and equipment
4: Protocol
5: Analysis
6: Optimization and troubleshooting
7: Conclusion
Acknowledgment
Chapter Seventeen: Leishmania tarentolae cell-free based approach for rapid anitbody–antigen interaction analysis
Abstract
1: Introduction
2: Materials
3: Methods
4: Summary
5: Notes
6: Protein sequences
Chapter Eighteen: Cell-free protein synthesis using Chinese hamster ovary cells
Abstract
1: Introduction
2: Key resources table
3: Materials and equipment
4: Step-by-step method details
5: Expected outcomes
6: Advantages
7: Limitations
8: Optimization and troubleshooting
9: Safety considerations and standards
10: Alternative methods/procedures