Protein Purification Applications A Practical Approach

by Roe, Simon

• ISBN13: 9780199636723
• ISBN10: 0199636729
• Edition: 2nd
• Format: Hardcover
• Copyright: 2001-04-05
• Publisher: Oxford University Press

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