Protein Purification Applications A Practical Approach 2nd Edition by Simon Roe 0199636710 9780199636716

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ISBN 10: 0199636710

ISBN 13: 9780199636716

Author: Simon Roe

Proteins are an integral part of molecular and cellular structure and function and are probably the most purified type of biological molecule. In order to elucidate the structure and function of any protein it is first necessary to purify it. Protein purification techniques have evolved over the past ten years with improvements in equipment control, automation, and separation materials, and the introduction of new techniques such as affinity membranes and expanded beds. These developments have reduced the workload involved in protein purification, but there is still a need to consider how unit operations linked together to form a purification strategy, which can be scaled up if necessary. The two Practical Approach books on protein purification have therefore been thoroughly updated and rewritten where necessary. The core of both books is the provision of detailed practical guidelines aimed particularly at laboratory scale purification. Information on scale-up considerations is given where appropriate. The books are not comprehensive but do cover the major laboratory techniques and common sources of protein. Protein Purification Techniques focuses on unit operations and analytical techniques. It starts with an overview of purification strategy and then covers initial extraction and clarification techniques. The rest of the book concentrates on different purification methods with the emphasis being on chromatography. The final chapter considers general scale-up considerations. Protein Purification Applications describes purification strategies from common sources: mammalian cell culture, microbial cell culture, milk, animal tissue, and plant tissue. It also includes chapters on purification of inclusion bodies, fusion proteins, and purification for crystallography. A purification strategy that can produce a highly pure single protein from a crude mixture of proteins, carbohydrates, lipids, and cell debris to is a work of art to be admired. These books (available individually or as a set)are designed to give the laboratory worker the information needed to undertake the challenge of designing such a strategy.

Protein Purification Applications A Practical Approach 2nd Table of contents:

1 Fusion protein purification methods

1 Introduction

2 Protecting proteins from proteolysis

Purification of fused APIII and release of protein using V8 protease

Isolation of EGF from tryp E-fusion using trypsin

3 Engineering to improve purification processes

Fluidized bed IMAC for polyHis-tagged proteins

Engineering metal affinity sites in somatotropins

Engineering proteins for polyelectrolyte precipitation

4 Fusions of generic application

PolyHistidine

FLAG

Recombinant phage antibody system

Fusion tag detection and purification: S-Tag system

T7-Tag affinity purification kit

Fusions based on maltose-binding protein

Intein-mediated purification with an affinity chitin binding tag

5 Concluding remarks

References

2 Initial purification of Inclusion bodies

1 Introduction

2 Mechanism of inclusion body formation

3 Isolation of inclusion bodies from cell homogenates

4 Washing of inclusion bodies

References

3 Purification for crystallography

1 Introduction

2 Methods for protein crystallization

Batch

Vapour diffusion

Dialysis

3 Influence of heterogeneity on crystallization

Effects of impurities

Origins of microheterogeneity

Detection of microheterogeneity

Additives and proteolysis

4 Concluding remarks

References

4 Protein purifications from mammalian cell culture

1 Introduction

2 Nature of recombinant proteins produced in mammalian cells

3 Transgenic animals

4 Overview of extraction and purification process

5 Process design—preventive measures

Cell culture system

Production cells

Culture medium

6 Cell separation

Purification without cell separation

7 Initial product recovery and fractionation

Recovery of recombinant protein from milk

Direct capture of product from feedstock

8 Main purification

Ion exchange chromatography

Hydrophobic interaction chromatography

Affinity chromatography systems

Small molecule affinity ligands

9 Particle-tolerant chromatographic supports

10 Purification of monoclonal antibodies

Separation using protein A

Monoclonal antibody purification by ion exchange

Other recent approaches to purification of monoclonal antibodies

11 The elimination of viral contamination risks

‘Spiking’ and the use of model viruses

Scale-down of unit operations

Calculation of virus clearance

A cautionary note

Virus removal by membrane filtration

12 Elimination of contaminating DNA

13 Other biologically active contaminants

14 Polishing steps

15 Problems

References

5 Protein purification from microbial cell culture

1 Introduction

2 Chromatographic processes

Positive or negative steps

Batch or column contactors

Gradient elution

3 Ion exchange protein purifications from microbial cell culture

Isolation of yeast enzymes

Isolation of amylase from Bacillus subtilis

Isolation of DNA-modifying enzymes

4 Ion exchange nucleic acid purification from microbial culture

References

6 Protein purification from milk

1 Introduction

2 Milk proteins

Micelle structure

Major milk protein groups

3 Factors to consider in preparation and handling milk

Source

Proteolysis

Solubility

Protein modification

Drying and storage

4 Fractionation of milk proteins

Fractionation of casein and whey

Fractionation of milk proteins by differential solubility

5 Chromatography of milk proteins

Ion exchange

Size exclusion chromatography

Affinity chromatography

Reverse-phase chromatography

Chromatofocusing

6 Identification and analysis of purity

Polyacrylamide gel electrophoresis

Isoelectric focusing

Capillary electrophoresis

Analytical chromatography

Mass spectrometry

7 Developments

Preparative electrophoresis

Microfiltration and ultrafiltration

Aqueous two-phase partitioning

Acknowledgements

References

7 Protein purification from animal tissue

1 Introduction

2 Choice of tissue

3 Tissue disruption

4 Prevention of unwanted proteolysis

5 Subcellular fractionation

6 Solubilization of membrane proteins

7 Example purifications of animal proteins

Purification of angiotensin converting enzyme

Purification of aminopeptidase P

Purification of fructose-1,6-biphosphatase

References

Appendix

8 Protein purification from plant sources

1 Introduction

2 Special considerations

Low protein concentration

Proteolysis

Inhibition and tanning

3 Source of material

Leaf

Stem

Roots

Seeds and storage organs

Fruits

Tissue cultures

Organelles

4 Direct and indirect strategies for identification of subsets of plant proteins

5 Examples of successful purification protocols: enzymes of carbohydrate metabolism

Calvin cycle 24

Polysaccharide synthases

6 Enzymes of secondary metabolism

Phenolic metabolism

Terpenoids

Alkaloids

7 Preparation for Edman protein sequencing

References

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