Recombinant protein expression : eukaryotic hosts / edited by Zvi Kelman and William B. O'Dell.

Format:

Book

Contributor:

O'Dell, William B., editor.

Kelman, Zvi, editor.

Series:

Issn

Language:

English

Subjects (All):

Recombinant proteins.

Enzymes.

Gene expression.

Physical Description:

1 online resource (384 pages)

Edition:

1st ed.

Place of Publication:

Amsterdam, Netherlands ; London, England : Elsevier, [2022]

Summary:

Recombinant Protein Expression, Part B, Volume 660 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.

Contents:

Intro

Recombinant Protein Expression: Eukaryotic Hosts

Copyright

Contents

Contributors

Preface

Yeast hosts

Baculovirus-insect cell systems

Plant hosts

Mammalian hosts

Section I: Yeast hosts

Chapter One: Expression of recombinant multi-protein complexes in Saccharomyces cerevisiae

1. Introduction

2. Yeast strain

3. Gal 1/10 promotor and plasmid series

4. Protein affinity tags

4.1. Sequences of common peptide tags

5. Induction of recombinant protein/complexes from an episomal plasmid(s)

5.1. Transformation of expression plasmid(s) into the host cell

5.1.1. Materials and reagents

5.1.2. Method of yeast transformation

5.2. Cell growth and induction

5.2.1. Materials and reagents

5.2.2. Method of cell growth and induction

5.3. Harvesting and storage of induced yeast cells

5.3.1. Materials and reagents

5.3.2. Protocol

5.4. Cell disruption to release proteins for purification

5.4.1. Materials and reagents

5.4.2. Protocol

6. Expression of genes that are integrated into the yeast genome

6.1. Materials and reagents

6.2. Protocol for gene integration

6.3. Induction, cell harvesting and cell disruption

7. Overview and conclusion

Acknowledgments

References

Chapter Two: Recombinant production of membrane proteins in yeast

2. The Saccharomyces cerevisiae expression platform

3. Initial screening for optimal expression and protein purification conditions

4. Generation of clean expression plasmids by homologous recombination

5. Application of yeast for in vivo measurement of recombinant membrane protein activity

6. Key resources table

7. Materials and equipment

7.1. Equipment

7.2. Materials

7.3. Solutions

7.3.1. Transformation and growth of S. cerevisiae

7.3.2. Transformation and growth of E. coli.

7.3.3. Membrane preparations

7.3.4. Detergents

7.3.5. FSEC and SEC

7.3.6. IMAC purification

8. Step-by-step method details

8.1. Protocol

8.2. Step 1 The expression plasmid will be generated directly in the yeast host strain

8.2.1. Overview

8.2.2. Duration

8.3. Step 2 In vivo assembly of the expression constructs directly in the PAP1500 yeast strain

8.3.1. Overview

8.3.2. Duration

8.4. Step 3 Screening for fluorescent clones

8.4.1. Overview

8.4.2. Duration

8.5. Step 4 Rescue of plasmid DNA into E. coli

8.5.1. Overview

8.5.2. Duration

8.6. Step 5 Transformation of yeast DNA into E. coli

8.6.1. Overview

8.6.2. Duration

8.7. Step 6 Small scale expression screening at 15C and 30C

8.7.1. Overview

8.7.2. Duration

8.8. Step 7 Small scale preparation of yeast membranes

8.8.1. Overview

8.8.2. Duration

8.9. Step 8 Visualization of the produced membrane protein-GFP fusion by in-gel fluorescence

8.9.1. Overview

8.9.2. Duration

8.10. Step 9 Screen to identify detergents with high solubilization efficiencies

8.10.1. Overview

8.10.2. Duration

8.11. Step 10 Fluorescence size-exclusion chromatography to visualize protein quality

8.11.1. Overview

8.11.2. Duration

8.12. Step 11 IMAC purification of detergent solubilized protein

8.12.1. Overview

8.12.2. Duration

8.13. Step 12 Production of His-tagged versions of the target protein

8.13.1. Overview

8.13.2. Duration

9. Expected outcomes

10. Advantages

11. Limitations

12. Optimization and troubleshooting

12.1. Low expression level and insufficient solubilization in mild detergents

12.2. Potential solution to optimize the procedure

13. Safety considerations and standards

14. Alternative methods/procedures

Chapter Three: Expression of proteins in Pichia pastoris.

1. Introduction

2. Before you begin

3. Key resources table

4. Materials and equipment

4.1. Reagents

4.2. Materials

4.3. Equipment

5. Step-by-step method details

5.1. Cloning and transforming into Pichia pastoris

5.2. Screening for high expressing clones

5.2.1. Protein analysis

5.2.2. LPTVA

5.3. Larger-scale expression

6. Expected outcomes

7. Quantification and statistical analysis

8. Advantages

9. Limitations

10. Optimization and troubleshooting

10.1. Genetic engineering strategies

10.2. Strain engineering strategies

10.3. Cultivation strategies

11. Safety considerations and standards

12. Alternative methods/procedures

13. Notes

Chapter Four: Hybrid-architectured promoter design to engineer expression in yeast

2. Hybrid-architectured promoter design to engineer recombinant protein expression

2.1. Hybrid-architectured EPV design concept

3. Method for hybrid-architectured EPV design

3.1. Promoter architecture and prediction of cis-acting DNA sequences and target TFs

3.1.1. Information systems for the analysis and prediction of transcription regulatory associations

3.1.2. Identification of putative TFBSs by in silico analysis and manual curation

3.2. Selection of synthetic cis-acting DNA sites for hybrid-architectured EPVs

3.2.1. Yeast alcohol dehydrogenase (ADH) promoters

3.2.2. ADH2 promoter architecture and prediction of cis-acting DNA sites

3.2.2.1. Sequence 1: Sequence of ADH2 promoter (PADH2) in P. pastoris

3.2.3. Selection of synthetic cis-acting DNA sites for EPVs of ADH2

3.3. Constraints and parameters in hybrid-architectured EPV design

3.3.1. Design and construction of hybrid-architectured ADH2 EPVs

4. Protocol

4.1. Equipment

4.2. Chemicals and recombinant proteins.

4.3. Critical commercial assays

4.4. Bacterial and yeast strains

4.5. List of primers

4.6. Protocol for construction of EPVs

4.6.1. Sequence 2: Nucleotide sequence of enhanced green fluorescent protein (eGFP) gene

4.7. Screening conditions and analytical methods

4.8. Statistical analysis

5. Summary

Chapter Five: Hybrid-architectured promoter design to deregulate expression in yeast

2. Hybrid-architectured engineered promoter design for deregulated expression in yeast

2.1. Hybrid-architectured engineered promoter variant (EPV) design concept

3. Method for hybrid-architectured EPV design to deregulate expression

3.1. Prediction of cis-acting DNA sequences and target transcription factors

3.1.2. Identification of putative TF binding sites by in silico analysis and manual curation

3.1.3. P. pastoris AOX1 promoter

3.2. Selection of synthetic cis-acting DNA sites for hybrid-architectured EPVs of AOX1

3.2.1. Determination of synthetic cis-acting DNA sites for deregulated AOX1 promoter expression

3.3. Constraints and parameters in hybrid-architectured EPV design for deregulation of expression

3.3.1. Design and construction of hybrid-architectured AOX1 EPVs for deregulation of expression

3.3.1.1. EPV designed for Mxr1 binding

3.3.1.1.1. Design of PAOX1/Adr1-L3

3.3.1.2. EPV designed for Cat8 binding

3.3.1.2.1. Design of PAOX1/Cat8-L3

3.3.1.3. EPV designed with two different synthetic TFBSs for Cat8 and Mxr1 binding

4.2. Chemicals and recombinant proteins

4.3. Critical commercial assays

4.5. List of primers.

4.6. Protocol for construction of EPVs for deregulation of expression

Acknowledgment

Section II: Baculovirus-insect cell systems

Chapter Six: The MultiBac BEVS: Basics, applications, performance and recent developments

2. MultiBac development

3. Baculovirus-vectored heterologous protein expression protocol

4. Key resources table

5. Materials and equipment

5.1. Other buffers and materials

5.2. Equipment

6. Basic step-by-step method for baculovirus-vectored heterologous protein expression

6.1. Before you begin

6.2. Blue/white screening and transfection

6.3. Virus amplification and protein expression test

7. Expected outcomes

10. Safety considerations and standards

11. Alternative methods/procedures

12. Expanding the scope

12.1. Mammalian transduction and DNA delivery

13. Use of MultiBac for SARS-CoV-2 research

14. Conclusions and outlook

Chapter Seven: Applications of Golden Gate cloning to protein production using the baculovirus expression vector system

2. Vector design

2.1. Design of the single tier GoldenBac vectors

2.2. Design of the two-tier ``Baustein´´ vectors

3. Protocol

3.1. Construction of GoldenBac expression constructs

3.1.1. Before you begin

3.1.2. Materials and equipment

3.1.3. Step 1-Golden Gate reaction

3.1.4. Step 2-Construct confirmation

3.2. Construction of Baustein expression constructs

3.2.1. Before you begin

3.2.2. Materials and equipment

3.2.3. Step 1-Golden Gate reaction with SapI

3.2.4. Step 2-Level 1 construct validation

3.2.5. Step 3-Golden Gate reaction with BsaI

3.2.6. Step 4-Level 2 construct validation

4. Advantages.

5. Limitations.

Notes:

Includes bibliographical references and index.

Description based on print version record.

Description based on publisher supplied metadata and other sources.

ISBN:

0-323-90738-5

OCLC:

1285170885