Protein Purification Techniques A Practical Approach

by Roe, Simon

• ISBN13: 9780199636747
• ISBN10: 0199636745
• 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
Purification strategy	1	(10)
Simon D. Roe
Key considerations	1	(1)
Aims of purification---why?	2	(1)
The target protein and contaminants	3	(1)
Protein structure	4	(1)
Source of the product	5	(1)
Key steps in purification	6	(1)
Process integration	6	(1)
Scale-up	7	(1)
Monitoring the purification---assays	8	(1)
Time and temperature	8	(3)
References	10	(1)
Getting started	11	(16)
Simon D. Roe
Overview of lab equipment	11	(3)
Ancillary equipment	12	(1)
Detection and analysis equipment	12	(1)
Seperations equipment	13	(1)
Control of pH---buffers---principles, selection, and use	14	(3)
Purification strategy	17	(6)
Ordering of steps	17	(1)
Nature of starting material	17	(1)
Storage of material	18	(1)
Fermentation products	19	(1)
Other sources of proteins	19	(1)
Making an extract	19	(2)
Separation of particulates	21	(1)
Protein concentration	22	(1)
Adjusting sample composition between steps	22	(1)
Protein lability and structure---implications for purification	23	(4)
Introduction	23	(1)
Denaturation	24	(1)
Catalytic site inactivation	24	(1)
Proteolysis	24	(1)
Other causes of yield loss	25	(1)
References	26	(1)
Analysis of purity	27	(24)
Dev Baines
Introduction	27	(1)
Considering yield and purity	27	(1)
Scope of the methods included	28	(1)
Total protein quantitation	28	(6)
Ultraviolet absorption protein assay	28	(2)
Lowry (Folin-Ciocalteau) protein assay	30	(1)
Bradford (Coomassie Brilliant Blue) dye-binding protein assay	31	(1)
Bicinchoninic acid protein assay	32	(1)
Colloidal gold protein assay	32	(1)
General comments	33	(1)
Specific quantitation	34	(4)
Activity assays	34	(2)
Binding assays	36	(2)
Detection of impurities	38	(11)
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis	38	(2)
Detection of protein bands in electrophoresis gels	40	(2)
Analysis by isoelectric focusing (IEF)	42	(2)
Glycoprotein analysis	44	(1)
Western blotting	45	(2)
Chromatographic methods	47	(2)
Other biochemical and biophysical methods	49	(2)
Protein mass spectrometry	49	(1)
References	49	(2)
Clarification techniques	51	(32)
Ian Reed
Duncan Mackay
Introduction	51	(1)
Sedimentation	52	(2)
Principles of sedimentation	52	(2)
Gravity sedimentation	54	(1)
Centrifugal sedimentation	54	(6)
Centrifuge equipment	55	(1)
Laboratory centrifuge test work	56	(3)
Suspension conditioning	59	(1)
Filtration	60	(5)
Background	60	(2)
Principles of dead-end filtration	62	(2)
Laboratory scale filtration equipment	64	(1)
Application of filtration	65	(1)
Membrane processes	65	(15)
Background	65	(2)
Principles of operation	67	(4)
Membrane equipment	71	(2)
Membrane selection	73	(3)
Membrane operation	76	(4)
Choice of clarification process	80	(3)
Cell disintegration and extraction techniques	83	(28)
R. H. Cumming
G. Iceton
Overview	83	(8)
Stability of the released protein	84	(1)
Location of target protein within the cell	84	(1)
The yield and kinetics of the process	85	(1)
Continuous or batch disruption	86	(1)
The need to consider subsequent steps	87	(1)
Assessing the extent of disruption	88	(1)
Marker substances for cell disruption	89	(1)
Containment of the process	90	(1)
Scale-up	91	(1)
Methods of disruption	91	(17)
Pre-treatment of material	91	(1)
General procedure notes	92	(1)
Mixers and blenders	92	(2)
Coarse grinding: pestle and mortar	94	(1)
Fine grinding: the bead mill	94	(2)
Homogenization	96	(3)
Ultrasonication	99	(3)
Heat shock	102	(1)
Freezing and thawing	102	(1)
Osmotic shock	103	(1)
Lytic enzymes	103	(2)
Chemical treatments	105	(1)
Detergents	105	(2)
Solvents	107	(1)
Conclusions: choice of methods	108	(3)
References	108	(3)
Concentration of the extract	111	(44)
E. L. V. Harris
Introduction	111	(1)
Addition of a dry matrix polymer	112	(1)
Ultrafiltration	112	(17)
Equipment	116	(8)
Operation	124	(3)
Other applications of ultrafiltration	127	(2)
Freeze-drying or lyophilization	129	(1)
Removal of salts and exchange of buffer	130	(2)
Dialysis	130	(2)
Diafiltration	132	(1)
Gel filtration	132	(1)
Purification and concentration by precipitation	132	(11)
Precipitation by alteration of the pH	134	(1)
Precipitation by decreasing the ionic strength	135	(1)
Precipitation by increasing the ionic strength (salting-out)	135	(4)
Precipitation by organic solvents	139	(2)
Precipitation by organic polymers	141	(1)
Precipitation by denaturation	141	(2)
Aqueous two-phase partitioning	143	(12)
B. A. Andrews
J. A. Asenjo
Equipment and materials	146	(1)
Optimization	146	(2)
Using phase partitioning in a purification procedure	148	(2)
Affinity partitioning	150	(2)
Acknowledgements	152	(1)
References	152	(3)
Separation on the basis of chemistry	155	(32)
Lars Hagel
Introduction	155	(1)
Chemical interaction between surfaces	155	(3)
Basic properties of proteins	158	(2)
Properties of the solvent	160	(1)
Properties of chromatography media	161	(26)
Ion exchange chromatography	166	(6)
Hydrophobic interaction chromatography	172	(3)
Reversed-phase chromatography	175	(2)
Charge-transfer chromatography	177	(2)
Covalent chromatography	179	(3)
Design, optimization, and scale-up of a chromatographic purification procedure	182	(1)
Acknowledgements	183	(1)
References	183	(4)
Chromatography on the basis of size	187	(26)
P. Cutler
Introduction	187	(10)
Principle	187	(5)
Applications of size exclusion chromatography	192	(5)
Materials	197	(5)
Equipment	197	(2)
Mobile phase	199	(2)
Non-ideal size exclusion	201	(1)
Methods	202	(11)
Flow rates	202	(1)
Preparation and packing	202	(2)
Equilibrium	204	(1)
Sample preparation and application	205	(1)
Evaluation and calibration	205	(2)
Separation of proteins	207	(1)
Column cleaning and storage	208	(1)
Trouble shooting	209	(1)
References	209	(4)
Purification by exploitation of activity	213	(28)
S. Angal
P. D. G. Dean
Simon D. Roe
Introduction	213	(1)
Design and preparation of affinity adsorbents	214	(9)
Choice of ligand	214	(1)
Selection of the matrix	215	(2)
Choice of spacer	217	(1)
Activation and coupling chemistry	218	(4)
Estimation of ligand concentration	222	(1)
Use of affinity adsorbents	223	(7)
Initial questions	223	(1)
Selection of conditions for operation	223	(3)
Estimation of capacity	226	(1)
Ligand efficiency	227	(1)
Application to protein purification	227	(3)
Immunopurification	230	(11)
C. R. Hill
L. G. Thompson
A. C. Kenney
Antibody selection	231	(1)
Immobilization of antibodies on CNBr-activated Sepharose	232	(3)
Procedures for immunopurification	235	(4)
References	239	(2)
Scale-up considerations	241	(14)
John B. Noble
Scale-up of purification processes and unit operations	241	(14)
Choice of scale	241	(1)
Overview of critical issues	242	(3)
Practical aspects of scale-up	245	(4)
Case study 1	249	(1)
Case study 2	250	(3)
References	253	(2)
A1 List of suppliers	255	(4)
Index	259