9780199636723

Protein Purification Applications A Practical Approach

by
  • ISBN13:

    9780199636723

  • ISBN10:

    0199636729

  • Edition: 2nd
  • Format: Hardcover
  • Copyright: 2001-04-05
  • Publisher: Oxford University Press

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Summary

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.

Table of Contents

List of protocols page
xiii
Abbreviations xv
Fusion protein purification methods
1(18)
Stephen J. Brewer
Charles E. Glatz
Charles Dickerson
Introduction
1(1)
Protecting proteins from proteolysis
2(5)
Purification of fused APIII and release of protein using V8 protease
4(1)
Isolation of EGF from tryp E-fusion using trypsin
5(2)
Engineering to improve purification processes
7(5)
Fluidized bed IMAC for polyHis-tagged proteins
8(2)
Engineering metal affinity sites in somatotropins
10(1)
Engineering proteins for polyelectrolyte precipitation
11(1)
Fusions of generic application
12(4)
PolyHistidine
13(1)
FLAG
14(1)
Recombinant phage antibody system
15(1)
Fusion tag detection and purification: S. Tag system
15(1)
T7.Tag affinity purification kit
15(1)
Fusions based on maltose-binding protein
16(1)
Intein-mediated purification with an affinity chitin binding tag
16(1)
Concluding remarks
16(3)
References
17(2)
Initial purification of inclusion bodies
19(10)
Bernard N. Violand
Introduction
19(1)
Mechanism of inclusion body formation
19(1)
Isolation of inclusion bodies from cell homogenates
20(2)
Washing of inclusion bodies
22(7)
References
26(3)
Purification for crystallography
29(20)
S.P. Wood
A.R. Coker
Introduction
29(4)
Methods for protein crystallization
33(5)
Batch
33(2)
Vapour diffusion
35(2)
Dialysis
37(1)
Influence of heterogeneity on crystallization
38(7)
Effects of impurities
38(1)
Origins of microheterogeneity
39(2)
Detection of microheterogeneity
41(2)
Additives and proteolysis
43(2)
Concluding remarks
45(4)
References
45(4)
Protein purifications from mammalian cell culture
49(24)
Terry Cartwright
Introduction
49(1)
Nature of recombinant proteins produced in mammalian cells
50(1)
Transgenic animals
51(1)
Overview of extraction and purification process
52(1)
Process design---preventive measures
53(2)
Cell culture system
53(1)
Production cells
54(1)
Culture medium
54(1)
Cell separation
55(1)
Purification without cell separation
56(1)
Initial product recovery and fractionation
56(1)
Recovery of recombinant protein from milk
57(1)
Direct capture of product from feedstock
57(1)
Main purification
57(4)
Ion exchange chromatography
58(1)
Hydrophobic interaction chromatography
58(1)
Affinity chromatography systems
59(1)
Small molecule affinity ligands
60(1)
Particle-tolerant chromatographic supports
61(1)
Purification of monoclonal antibodies
62(4)
Separation using protein A
62(2)
Monoclonal antibody purification by ion exchange
64(1)
Other recent approaches to purification of monoclonal antibodies
65(1)
The elimination of viral contamination risks
66(3)
`Spiking' and the use of model viruses
66(1)
Scale-down of unit operations
67(1)
Calculation of virus clearance
68(1)
A cautionary note
68(1)
Virus removal by membrane filtration
69(1)
Elimination of contaminating DNA
69(1)
Other biologically active contaminants
70(1)
Polishing steps
70(1)
Problems
70(3)
References
71(2)
Protein purification from microbial cell culture
73(14)
Peter R. Levison
Introduction
73(1)
Chromatographic processes
73(3)
Positive or negative steps
74(1)
Batch or column contactors
75(1)
Gradient elution
75(1)
Ion exchange protein purifications from microbial cell culture
76(7)
Isolation of yeast enzymes
76(3)
Isolation of amylase from Bacillus subtilis
79(2)
Isolation of DNA-modifying enzymes
81(2)
Ion exchange nucleic acid purification from microbial culture
83(4)
References
85(2)
Protein purification from milk
87(30)
Richard Burr
Introduction
87(1)
Milk proteins
87(6)
Micelle structure
88(1)
Major milk protein groups
88(5)
Factors to consider in preparation and handling milk
93(2)
Source
93(1)
Proteolysis
93(1)
Solubility
93(1)
Protein modification
94(1)
Drying and storage
95(1)
Fractionation of milk proteins
95(6)
Fractionation of casein and whey
95(5)
Fractionation of milk proteins by differential solubility
100(1)
Chromatography of milk proteins
101(5)
Ion exchange
101(2)
Size exclusion chromatography
103(1)
Affinity chromatography
104(1)
Reverse-phase chromatography
105(1)
Chromatofocusing
105(1)
Identification and analysis of purity
106(4)
Polyacrylamide gel electrophoresis
106(3)
Isoelectric focusing
109(1)
Capillary electrophoresis
110(1)
Analytical chromatography
110(1)
Mass spectrometry
110(1)
Developments
110(7)
Preparative electrophoresis
110(1)
Microfiltration and ultrafiltration
111(1)
Aqueous two-phase partitioning
111(1)
Acknowledgements
112(1)
References
112(5)
Protein purification from animal tissue
117(18)
Nigel M. Hooper
Introduction
117(1)
Choice of tissue
117(1)
Tissue disruption
118(1)
Prevention of unwanted proteolysis
119(1)
Subcellular fractionation
119(5)
Solubilization of membrane proteins
124(2)
Example purifications of animal proteins
126(9)
Purification of angiotensin converting enzyme
126(3)
Purification of aminopeptidase P
129(1)
Purification of fructose-1,6-biphosphatase
129(3)
References
132(1)
Appendix
132(3)
Protein purification from plant sources
135(24)
G. Paul Bolwell
Introduction
135(1)
Special considerations
136(2)
Low protein concentration
136(1)
Proteolysis
137(1)
Inhibition and tanning
138(1)
Source of material
138(9)
Leaf
138(2)
Stem
140(1)
Roots
140(1)
Seeds and storage organs
140(1)
Fruits
140(1)
Tissue cultures
141(1)
Organelles
141(6)
Direct and indirect strategies for identification of subsets of plant proteins
147(2)
Examples of successful purification protocols: enzymes of carbohydrate metabolism
149(3)
Calvin cycle 24
150(1)
Polysaccharide synthases
150(2)
Enzymes of secondary metabolism
152(3)
Phenolic metabolism
152(2)
Terpenoids
154(1)
Alkaloids
154(1)
Preparation for Edman protein sequencing
155(4)
References
156(3)
A1 List of suppliers 159(4)
Index 163

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