Protein expression: a practical approach
Gespeichert in:
| Format: | Buch |
|---|---|
| Sprache: | English |
| Veröffentlicht: |
Oxford [u.a.]
Oxford Univ. Press
1999
|
| Schriftenreihe: | The practical approach series
202 |
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | XIX, 282 S. Ill., graph. Darst. |
| ISBN: | 0199636230 0199636249 |
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| 245 | 1 | 0 | |a Protein expression |b a practical approach |c ed. by S. J. Higgins ... |
| 264 | 1 | |a Oxford [u.a.] |b Oxford Univ. Press |c 1999 | |
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Datensatz im Suchindex
| _version_ | 1804127080348123136 |
|---|---|
| adam_text | Contents
List of con trib u tors xv
Abbreviations xvii
1. Protein expression in mammalian cells 1
Marlies Otter Nilsson and Tommy Nilsson
1. Introduction 1
2. Viral and plasmid vectors 1
Semliki forest virus 1
Vaccinia virus 4
Retroviral vectors 7
Plasmid pCMUIV 8
Plasmid pSRa 10
3. Transient and stable transfection methods 10
Calcium phosphate 10
DEAE dextran 13
Lipid mediated transfection 14
Electroporation 15
Microinjection 16
Stable transfection and selection 18
Inducible protein expression in stable cell lines 20
4. Detection of expressed protein 22
GFP as a tool in protein expression 22
Epitope tags 23
References 25
2. Expression in Xenopus oocytes and
cell free extracts 29
Glenn M. Matthews
1. Introduction 29
Translation in oocytes 29
Xenopus egg extracts 30
Maintaining Xenopus laevis stocks 30
2. Xenopus oocyte microinjection 31
Equipment 31
Obtaining and culturing oocytes 32
mRNA 36
Contents
Use of the injector 38
Microinjection 40
Expression from microinjected DNA 41
Radioactive labelling 41
Analysis of radioactive translation products 43
Fractionation of oocytes 45
3. Preparation and use of Xenopus egg cell free extracts 46
Equipment 46
Xenopus eggs 48
Preparation of extract 49
In vitro translation using Xenopus cell free extracts 53
Analysis of translation products 55
References 58
3. Expressing cloned genes in the yeasts
Saccharomyces cerevisiae and
Pichia pastoris 6i
Mick F. Take, JeffJ. Clare, and Mike A. Romanos
1. Introduction 61
2. Saccharomyces cerevisiae expression systems 63
Plasmid based vectors 63
Transformation of S. cerevisiae 66
Choice of strain of S. cerevisiae 68
Transcription and translation of heterologous genes and cDNAs 69
Directing the extracellular synthesis of heterologous proteins 72
Analysis of heterologous gene expression 76
3. Pichia pastoris expression systems 83
Introduction 83
Expression strategies 84
Host vector systems and transformation methods for P. pastoris 87
Analysis of DNA from P. pastoris transformants 93
Induction of foreign protein expression in P. pastoris 95
References 99
4. Baculovirus expression systems 101
Claire L. Merrington, Linda A. King, and Robert D. Possee
1. Introduction 101
2. Baculovirus life cycle 102
3. Insect cell culture 103
x
Contents
4. Baculovirus expression vectors 109
Manipulating the baculovirus genome 109
Baculovirus transfer vectors 109
Preparation of recombinant transfer vectors 112
5. Preparation of recombinant virus 113
Optimizing the selection of recombinant virus 113
Co transfection of insect cells with linearized viral DNA and
recombinant transfer vectors 115
Identification and purification of recombinant viruses 117
6. Characterization of recombinant virus DNA 118
7. Analysis of protein synthesis in virus infected cells 120
8. Post translational modification of proteins synthesized
using the baculovirus expression system 122
9. Scaling up recombinant protein production 123
10. Alternative methods for producing recombinant
baculoviruses 124
Baculovirus yeast system 124
Bacmid system 125
11. Future developments of the baculovirus expression system 125
References 125
5. Protein synthesis in eukaryotic cell free
systems 129
Mike J. Clemens and Ger J. M. Pruijn
1. Introduction 129
2. Preparation of messenger RNAs 130
Precautions against RNase mediated degradation 130
Preparation of intact RNA from ribosomal and polysomal fractions 131
Oligo(dT) affinity chromatography for isolation of poly( A)+ mRNA 133
Isolation of individual mRN A species 133
Transcription of mRN A in vitro 134
3. The reticulocyte lysate cell free translation system 136
Preparation and storage of reticulocyte lysate 136
Assays of protein synthesis in reticulocyte lysates 140
Advantages and disadvantages of the reticulocyte lysate system 145
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
xi
Contents
5. Cell free translation systems from other eukaryotic cell types 150
6. Methods for analysis of translation products 151
Radioisotopic methods 151
Chemiluminescence 152
Immunoprecipitation of translation products 153
Ligand binding assays 155
In vitro synthesis of membrane and secretory proteins 155
7. Specialized procedures 156
Synthesis of biotinylated proteins 156
Coupled in vitro transcription translation systems 157
Cap dependent versus internal initiation of translation 157
Assays for post translational processing 158
The protein truncation test 165
Acknowledgements 165
References 165
6. Prokaryotic in vivo expression systems 169
Edward R. Appelbaum and Allan R. Shatzman
1. Introduction 169
2. General considerations in selecting an E. coli expression
system 169
Choosing between E. coli and other expression systems 169
Improving the level of expression 171
Improving the solubility of a protein expressed in E. coli 172
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. coli expression systems 175
Promoters and other transcription regulatory elements 175
Translation initiation and termination signals 177
Host strain 178
4. Protocols for expression: general comments 178
5. Expression, detection, and purification of a His6 tagged
protein 180
Construction of the recombinant vector and transformation of
host cells 180
Expression of the heterologous sequence 187
Analysis of expression of the heterologous protein 189
Purification of His6 tagged proteins 193
6. Expression of heterologous proteins in a secretion system 196
7. Sources of information on expression systems 199
xii
Contents
Acknowledgements 199
References 199
7. Cell free coupled transcription translation
systems from Escherichia coli 201
Gisela Kramer, Wieslaw Kudlicki, and Boyd Hardesty
1. Background 201
Bacterial cell free expression systems 201
Usefulness of cell free coupled transcription translation systems 202
2. Preparation of extracts and components for coupled
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 coupled
transcription translation 210
3. The coupled transcription translation assay 211
The basic assay 211
Analysis of the product formed in the coupled transcription
translation assay 213
4. Modified coupled transcription translation assays 219
5. Further developments 221
Acknowledgements 222
References 222
8. Monitoring protein expression 225
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 230
Western blotting 232
Immunodetection of proteins on dot blots and Western blots 238
Pulse chase labelling and immunoprecipitation of proteins 247
Examination of protein expression by immunomicroscopy 254
3. Monitoring of protein expression by epitope tagging 257
xiii
Contents
4. Surrogate reporter systems for monitoring protein
expression 259
General principles 259
Quantification of protein X expression using the CAT reporter
assay 260
Histological examination of protein expression using GUS reporter
activity 262
Monitoring expression and cellular location using GFP 262
Acknowledgements 264
References 264
Appendix i i
Index 273
xiv
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| spelling | Protein expression a practical approach ed. by S. J. Higgins ... Oxford [u.a.] Oxford Univ. Press 1999 XIX, 282 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier The practical approach series 202 Protéines - Synthèse - Manuels de laboratoire ram Protéines - Synthèse Gene Expression Regulation Laboratory Manuals Genetic translation Laboratory manuals Protein Biosynthesis Laboratory Manuals Proteins Synthesis Laboratory manuals Proteins chemical synthesis Laboratory Manuals Genexpression (DE-588)4020136-3 gnd rswk-swf Methode (DE-588)4038971-6 gnd rswk-swf (DE-588)4143413-4 Aufsatzsammlung gnd-content Genexpression (DE-588)4020136-3 s DE-604 Methode (DE-588)4038971-6 s Higgins, Stephen J. Sonstige oth The practical approach series 202 (DE-604)BV007921321 202 HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=008443157&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Protein expression a practical approach The practical approach series Protéines - Synthèse - Manuels de laboratoire ram Protéines - Synthèse Gene Expression Regulation Laboratory Manuals Genetic translation Laboratory manuals Protein Biosynthesis Laboratory Manuals Proteins Synthesis Laboratory manuals Proteins chemical synthesis Laboratory Manuals Genexpression (DE-588)4020136-3 gnd Methode (DE-588)4038971-6 gnd |
| subject_GND | (DE-588)4020136-3 (DE-588)4038971-6 (DE-588)4143413-4 |
| title | Protein expression a practical approach |
| title_auth | Protein expression a practical approach |
| title_exact_search | Protein expression a practical approach |
| title_full | Protein expression a practical approach ed. by S. J. Higgins ... |
| title_fullStr | Protein expression a practical approach ed. by S. J. Higgins ... |
| title_full_unstemmed | Protein expression a practical approach ed. by S. J. Higgins ... |
| title_short | Protein expression |
| title_sort | protein expression a practical approach |
| title_sub | a practical approach |
| topic | Protéines - Synthèse - Manuels de laboratoire ram Protéines - Synthèse Gene Expression Regulation Laboratory Manuals Genetic translation Laboratory manuals Protein Biosynthesis Laboratory Manuals Proteins Synthesis Laboratory manuals Proteins chemical synthesis Laboratory Manuals Genexpression (DE-588)4020136-3 gnd Methode (DE-588)4038971-6 gnd |
| topic_facet | Protéines - Synthèse - Manuels de laboratoire Protéines - Synthèse Gene Expression Regulation Laboratory Manuals Genetic translation Laboratory manuals Protein Biosynthesis Laboratory Manuals Proteins Synthesis Laboratory manuals Proteins chemical synthesis Laboratory Manuals Genexpression Methode Aufsatzsammlung |
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