Protein Expression A Practical Approach,9780199636242

Protein Expression A Practical Approach

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Edition: 1st
ISBN13: 9780199636242
ISBN10: 0199636249
Format: Hardcover
Pub. Date: 1999-07-29
Publisher(s): Oxford University Press
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Summary

Protein Expression: A Practical Approach and its companion volume Post-translational Modification: A Practical Approach complete the mini-series of Practical Approach books covering the synthesis and subsequent processing of proteins. Protein Expression: A Practical Approach details theexpression of cloned DNA or RNA templates in all the major in vivo and in vitro systems. The in vivo systems covered are cultured mammalian cells, the yeasts Saccharomyces cerevisiae and Pichia pastoris, baculovirus, Xenopus oocytes, and prokaryotic cells. Cell-free systems of both eukarytotes andprokaryotes are described, including the prokaryotic systems that offer copuled transcription- translation. There is also a chapter on monitoring protein expression. The post- translational fate of proteins is covered in Post-Translational Processing: A Practical Approach.

Table of Contents

List of contributors
xv
Abbreviations xvii
Protein expression in mammalian cells
1(28)
Marlies Otter-Nilsson
Tommy Nilsson
Introduction
1(1)
Viral and plasmid vectors
1(9)
Semliki forest virus
1(3)
Vaccinia virus
4(3)
Retroviral vectors
7(1)
Plasmid pCMUIV
8(2)
Plasmid pSRα
10(1)
Transient and stable transfection methods
10(12)
Calcium phosphate
10(3)
DEAE--dextran
13(1)
Lipid-mediated transfection
14(1)
Electroporation
15(1)
Microinjection
16(2)
Stable transfection and selection
18(2)
Inducible protein expression in stable cell lines
20(2)
Detection of expressed protein
22(7)
GFP as a tool in protein expression
22(1)
Epitope tags
23(2)
References
25(4)
Expression in Xenopus oocytes and cell-free extracts
29(32)
Glenn M. Matthews
Introduction
29(2)
Translation in oocytes
29(1)
Xenopus egg extracts
30(1)
Maintaining Xenopus laevis stocks
30(1)
Xenopus oocyte microinjection
31(15)
Equipment
31(1)
Obtaining and culturing oocytes
32(4)
mRNA
36(2)
Use of the injector
38(2)
Microinjection
40(1)
Expression from microinjected DNA
41(1)
Radioactive labelling
41(2)
Analysis of radioactive translation products
43(2)
Fractionation of oocytes
45(1)
Preparation and use of Xenopus egg cell-free extracts
46(15)
Equipment
46(2)
Xenopus eggs
48(1)
Preparation of extract
49(4)
In vitro translation using Xenopus cell-free extracts
53(2)
Analysis of translation products
55(3)
References
58(3)
Expressing cloned genes in the yeasts Saccharomyces cerevisiae and Pichia pastoris
61(40)
Mick F. Tuite
Jeff J. Clare
Mike A. Romanos
Introduction
61(2)
Saccharomyces cerevisiae expression systems
63(20)
Plasmid-based vectors
63(3)
Transformation of S. cerevisiae
66(2)
Choice of strain of S. cerevisiae
68(1)
Transcription and translation of heterologous genes and cDNAs
69(3)
Directing the extracellular synthesis of heterologous proteins
72(4)
Analysis of heterologous gene expression
76(7)
Pichia pastoris expression systems
83(18)
Introduction
83(1)
Expression strategies
84(3)
Host--vector systems and transformation methods for P. pastoris
87(6)
Analysis of DNA from P. pastoris transformants
93(2)
Induction of foreign protein expression in P. pastoris
95(4)
References
99(2)
Baculovirus expression systems
101(28)
Claire L. Merrington
Linda A. King
Robert D. Possee
Introduction
101(1)
Baculovirus life cycle
102(1)
Insect cell culture
103(6)
Baculovirus expression vectors
109(4)
Manipulating the baculovirus genome
109(1)
Baculovirus transfer vectors
109(3)
Preparation of recombinant transfer vectors
112(1)
Preparation of recombinant virus
113(5)
Optimizing the selection of recombinant virus
113(2)
Co-transfection of insect cells with linearized viral DNA and recombinant transfer vectors
115(2)
Identification and purification of recombinant viruses
117(1)
Characterization of recombinant virus DNA
118(2)
Analysis of protein synthesis in virus-infected cells
120(2)
Post-translational modification of proteins synthesized using the baculovirus expression system
122(1)
Scaling up recombinant protein production
123(1)
Alternative methods for producing recombinant baculoviruses
124(1)
Baculovirus--yeast system
124(1)
Bacmid system
125(1)
Future developments of the baculovirus expression system
125(4)
References
125(4)
Protein synthesis in eukaryotic cell-free systems
129(40)
Mike J. Clemens
Ger J. M. Pruijn
Introduction
129(1)
Preparation of messenger RNAs
130(6)
Precautions against RNase-mediated degradation
130(1)
Preparation of intact RNA from ribosomal and polysomal fractions
131(2)
Oligo(dT) affinity chromatography for isolation of poly(A)+ mRNA
133(1)
Isolation of individual mRNA species
133(1)
Transcription of mRNA in vitro
134(2)
The reticulocyte lysate cell-free translation system
136(10)
Preparation and storage of reticulocyte lysate
136(4)
Assays of protein synthesis in reticulocyte lysates
140(5)
Advantages and disadvantages of the reticulocyte lysate system
145(1)
The wheat germ cell-free translation system
146(4)
Sources of wheat germ
147(1)
Preparation of wheat germ extracts
147(1)
Assays of protein synthesis in wheat germ extracts
148(1)
Advantages and disadvantages of the wheat germ system
149(1)
Cell-free translation systems from other eukaryotic cell types
150(1)
Methods for analysis of translation products
151(5)
Radiosotopic methods
151(1)
Chemiluminescence
152(1)
Immunoprecipitation of translation products
153(2)
Ligand binding assays
155(1)
In vitro synthesis of membrane and secretory proteins
155(1)
Specialized procedures
156(13)
Synthesis of biotinylated proteins
156(1)
Coupled in vitro transcription--translation systems
157(1)
Cap-dependent versus internal initiation of translation
157(1)
Assays for post-translational processing
158(7)
The protein truncation test
165(1)
Acknowledgements
165(1)
References
165(4)
Prokaryotic in vivo expression systems
169(32)
Edward R. Appelbaum
Allan R. Shatzman
Introduction
169(1)
General considerations in selecting an E. coli expression system
169(6)
Choosing between E. coli and other expression systems
169(2)
Improving the level of expression
171(1)
Improving the solubility of a protein expressed in E. coli
172(2)
Expression of heterologous proteins as fusion proteins or with protein tags
174(1)
Nature of the N-terminus of the heterologous protein
175(1)
Features of E. coli expression systems
175(3)
Promoters and other transcription regulatory elements
175(2)
Translation initiation and termination signals
177(1)
Host strain
178(1)
Protocols for expression: general comments
178(2)
Expression, detection, and purification of a His6-tagged protein
180(16)
Construction of the recombinant vector and transformation of host cells
180(7)
Expression of the heterologous sequence
187(2)
Analysis of expression of the heterologous protein
189(4)
Purification of His6-tagged proteins
193(3)
Expression of heterologous proteins in a secretion system
196(3)
Sources of information on expression systems
199(2)
Acknowledgements
199(1)
References
199(2)
Cell-free coupled transcription-translation systems from Escherichia coli
201(24)
Gisela Kramer
Wieslaw Kudlicki
Boyd Hardesty
Background
201(2)
Bacterial cell-free expression systems
201(1)
Usefulness of cell-free coupled transcription--translation systems
202(1)
Preparation of extracts and components for coupled transcription--translation systems
203(8)
Preparation of the bacterial cell-free extract (S30)
203(2)
Construction and preparation of plasmids
205(3)
Preparation of RNA polymerases
208(2)
Preparation of the low molecular weight mix (LM) for coupled transcription--translation
210(1)
The coupled transcription--translation assay
211(8)
The basic assay
211(2)
Analysis of the product formed in the coupled transcription-translation assay
213(6)
Modified coupled transcription-translation assays
219(2)
Further developments
221(4)
Acknowledgements
222(1)
References
222(3)
Monitoring protein expression
225(42)
John Colyer
Introduction
225(2)
General considerations
225(1)
Basic strategies for monitoring protein expression
226(1)
Immunodetection of protein expression
227(30)
Considerations affecting the choice of antibody
227(3)
Immunodot blots
230(2)
Western blotting
232(6)
Immunodetection of proteins on dot blots and Western blots
238(9)
Pulse-chase labelling and immunoprecipitation of proteins
247(7)
Examination of protein expression by immunomicroscopy
254(3)
Monitoring of protein expression by epitope tagging
257(2)
Surrogate reporter systems for monitoring protein expression
259(8)
General principles
259(1)
Quantification of protein X expression using the CAT reporter assay
260(2)
Histological examination of protein expression using GUS reporter activity
262(1)
Monitoring expression and cellular location using GFP
262(2)
Acknowledgements
264(1)
References
264(3)
Appendix 267(6)
Index 273

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