Protein Expressio n
A Practical Approach
S . J . HIGGIN S
B . D . HAMES
List of contributors
xv
Abbreviations
Xvi i
1. Protein expression in mammalian cell s
Marlies Otter-Nilsson and Tommy Nilsso n
1. Introduction
1 2. Viral and plasmid vectors
1
Semliki forest virus
1 Vaccinia virus
4 Retroviral vectors
7 Plasmid pCMUIV
8 Plasmid pSRet
1 0
3. Transient and stable transfection methods
1 0 Calcium phosphate
1 0 DEAE-dextran
1 3 Lipid-mediated transfection
1 4 Electroporation
1 5 Microinjection
1 6 Stable transfection and selection
1 8 Inducible protein expression in stable cell lines
20
4. Detection of expressed protein
2 2 GFP as a tool in protein expression
2 2 Epitope tags
2 3
References
2 5
2. Expression in Xenopus oocytes an d cell-free extracts
2 9 Glenn M. Matthews
1. Introduction
29 Translation in oocytes
29 Xenopus egg extracts
30 Maintaining Xenopus laevis stocks
3 0
2. Xenopus oocyte microinjection
3 1 Equipment
3 1 Obtaining and culturing oocytes
3 2 mRNA
36
Analysis of radioactive translation products 43
Fractionation of oocytes 45
3 . Preparation and use ofXenopusegg cell-free extracts 46
Equipment 46
Xenopuseggs 48
Preparation of extract 49
In vitrotranslation usingXenopuscell-free extracts 5 3
Analysis of translation products 5 5
References 5 8
3 . Expressing cloned genes in the yeast s Saccharomyces cerevisiae an d
Pichia pastoris 6 1
Mick F. Tuite, Jeff J. Clare, and Mike A . Romanos
1. Introduction 6 1
2. Saccharomyces cerevisiae
expression systems 6 3Plasmid-based vectors 6 3
Transformation of S. cerevisiae 6 6
Choice of strain ofS. cerevisiae 6 8
Transcription and translation of heterologous genes and cDNAs 6 9 Directing the extracellular synthesis of heterologous proteins 7 2
Analysis of heterologous gene expression 7 6
3. Pichia pastoris
expression systems 8 3Introduction 8 3
Expression strategies 8 4
Host-vector systems and transformation methods for P. pastoris 8 7 Analysis of DNA fromP. pastoristransformants 9 3 Induction of foreign protein expression inP. pastoris 9 5
References 9 9
4. Baculovirus expression systems
10 1Claire L . Merrington, Linda A . King, and Robert D . Posse e
1. Introduction 10 1
2. Baculovirus life cycle 10 2
3. Insect cell culture 103
4. Baculovirus expression vectors
109
Manipulating the baculovirus genome
109 Baculovirus transfer vectors
109 Preparation of recombinant transfer vectors
11 2
5. Preparation of recombinant virus
11 3 Optimizing the selection of recombinant virus
11 3 Co-transfection of insect cells with linearized viral DNA an d
recombinant transfer vectors
11 5 Identification and purification of recombinant viruses
11 7
6. Characterization of recombinant virus DNA
11 8
7. Analysis of protein synthesis in virus-infected cells
12 0
8. Post-translational modification of proteins synthesize d using the baculovirus expression system
122
9. Scaling up recombinant protein production
123
10. Alternative methods for producing recombinan t baculoviruses
124 Baculovirus-yeast system
124 Bacmid system
125
11. Future developments of the baculovirus expression system
125References
125
5. Protein synthesis in eukaryotic cell-free systems
12 9
Mike J. Clemens and
GerJ. M. Pruijn
1. Introduction
129
2. Preparation of messenger RNAs
130 Precautions against RNase-mediated degradation 13 0 Preparation of intact RNA from ribosomal and polysomal fractions 13 1 Oligo(dT) affinity chromatography for isolation of poly(A) + mRNA 13 3 Isolation of individual mRNA species
13 3 Transcription of mRNA in vitro
13 4
3. The reticulocyte lysate cell-free translation system
13 6 Preparation and storage of reticulocyte lysate
13 6 Assays of protein synthesis in reticulocyte lysates
14 0 Advantages and disadvantages of the reticulocyte lysate system
14 5
4. The wheat germ cell-free translation system
146 Sources of wheat germ
147 Preparation of wheat germ extracts
147 Assays of protein synthesis in wheat germ extracts
148 Advantages and disadvantages of the wheat germ system
149
Chemiluminescence 15 2
Immunoprecipitation of translation products 15 3
Ligand binding assays 15 5
In vitrosynthesis of membrane and secretory proteins 15 5
7. Specialized procedures
15 6Synthesis of biotinylated proteins 15 6
Coupledin vitrotranscription-translation systems 15 7 Cap-dependent versus internal initiation of translation 15 7
Assays for post-translational processing 15 8
The protein truncation test 16 5
Acknowledgements
16 5References
16 56 . Prokaryotic in vivo expression systems
16 9Edward R . Appelbaum and Allan R . Shatzman
1. Introduction
1692. General considerations in selecting an
E . coliexpressio n
system
169Choosing betweenE. coliand other expression systems 169
Improving the level of expression 17 1
Improving the solubility of a protein expressed in E. coli 17 2 Expression of heterologous proteins as fusion proteins or with
protein tags 174
Nature of the N-terminus of the heterologous protein 175
3. Features of
E. coliexpression systems
17 5 Promoters and other transcription regulatory elements 175Translation initiation and termination signals 177
Host strain 178
4. Protocols for expression : general comments
17 85. Expression, detection, and purification of a Hisc tagge d
protein
180Construction of the recombinant vector and transformation o f
host cells 180
Expression of the heterologous sequence 187
Analysis of expression of the heterologous protein 189
Purification of His 6-tagged proteins 19 3
6. Expression of heterologous proteins in a secretion system
19 67. Sources of information on expression systems
199Acknowledgements
199
References
199
7 . Cell-free coupled transcription-translation systems from Escherichia coli
20 1 Gisela Kramer, Wieslaw Kudlicki, and Boyd Hardesty
1. Background
20 1 Bacterial cell-free expression systems
20 1 Usefulness of cell-free coupled transcription-translation systems
20 2
2. Preparation of extracts and components for couple d transcription-translation systems
203 Preparation of the bacterial cell-free extract (S30)
203 Construction and preparation of plasmids
205 Preparation of RNA polymerases
208 Preparation of the low molecular weight mix (LM) for couple d
transcription-translation
21 0
3. The coupled transcription-translation assay
21 1 The basic assay
21 1 Analysis of the product formed in the coupled transcription -
translation assay
21 3
4. Modified coupled transcription-translation assays
21 9
5. Further developments
221
Acknowledgements
22 2
References
22 2
8. Monitoring protein expression
22 5 John Colyer
1. Introduction
225 General considerations
225 Basic strategies for monitoring protein expression
226
2. Immunodetection of protein expression
227 Considerations affecting the choice of antibody
227 Immunodot blots
23 0 Western blotting
23 2 Immunodetection of proteins on dot blots and Western blots
23 8 Pulse-chase labelling and immunoprecipitation of proteins
24 7 Examination of protein expression by immunomicroscopy
25 4
3. Monitoring of protein expression by epitope tagging
257
Quantification of protein X expression using the CAT reporte r
assay 26 0
Histological examination of protein expression using GUS reporte r
activity 262
Monitoring expression and cellular location using GFP 26 2
