Protein Expression. A Practical Approach J. HIGGIN S

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1 Protein Expression A Practical Approach S. J. HIGGIN S B. D. HAMES

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3 List of contributors Abbreviations xv 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 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 Detection of expressed protein 2 2 GFP as a tool in protein expression 2 2 Epitope tags 2 3 References Expression in Xenopus oocytes and cell-free extracts 2 9 Glenn M. Matthews 1. Introduction 29 Translation in oocytes 29 Xenopus egg extracts 30 Maintaining Xenopus laevis stocks Xenopus oocyte microinjection 3 1 Equipment 3 1 Obtaining and culturing oocytes 3 2 mrna 36

4 Use of the injector 3 8 Microinjection 40 Expression from microinjected DNA 41 Radioactive labelling 41 Analysis of radioactive translation products 43 Fractionation of oocytes 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 5 3 Analysis of translation products 5 5 References 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 Saccharomyces cerevisiae expression systems 6 3 Plasmid-based vectors 6 3 Transformation of S. cerevisiae 6 6 Choice of strain of S. 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 Pichia pastoris expression systems 8 3 Introduction 8 3 Expression strategies 8 4 Host-vector systems and transformation methods for P. pastoris 8 7 Analysis of DNA from P. pastoris transformants 9 3 Induction of foreign protein expression in P. pastoris 9 5 References Baculovirus expression systems 10 1 Claire L. Merrington, Linda A. King, and Robert D. Posse e 1. Introduction Baculovirus life cycle Insect cell culture 103

5 4. Baculovirus expression vectors 109 Manipulating the baculovirus genome 109 Baculovirus transfer vectors 109 Preparation of recombinant transfer vectors 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 Characterization of recombinant virus DNA Analysis of protein synthesis in virus-infected cells Post-translational modification of proteins synthesize d using the baculovirus expression system Scaling up recombinant protein production Alternative methods for producing recombinan t baculoviruses 124 Baculovirus-yeast system 124 Bacmid system Future developments of the baculovirus expression system 125 References Protein synthesis in eukaryotic cell-free systems 12 9 Mike J. Clemens and Ger J. M. Pruijn 1. Introduction 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 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 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

6 5. Cell-free translation systems from other eukaryotic cell types Methods for analysis of translation products 15 1 Radioisotopic methods 15 1 Chemiluminescence 15 2 Immunoprecipitation of translation products 15 3 Ligand binding assays 15 5 In vitro synthesis of membrane and secretory proteins Specialized procedures 15 6 Synthesis of biotinylated proteins 15 6 Coupled in vitro transcription-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 5 References Prokaryotic in vivo expression systems 16 9 Edward R. Appelbaum and Allan R. Shatzman 1. Introduction General considerations in selecting an E. coli expressio n system 169 Choosing between E. coli and 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 Features of E. coli expression systems 17 5 Promoters and other transcription regulatory elements 175 Translation initiation and termination signals 177 Host strain Protocols for expression: general comments Expression, detection, and purification of a Hisc tagge d protein 180 Construction 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 Expression of heterologous proteins in a secretion system Sources of information on expression systems 199

7 Acknowledgements 199 References 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 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 The coupled transcription-translation assay 21 1 The basic assay 21 1 Analysis of the product formed in the coupled transcriptiontranslation assay Modified coupled transcription-translation assays Further developments 221 Acknowledgements 22 2 References Monitoring protein expression 22 5 John Colyer 1. Introduction 225 General considerations 225 Basic strategies for monitoring protein expression 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 Monitoring of protein expression by epitope tagging 257

8 4. Surrogate reporter systems for monitoring protein expression 25 9 General principles 25 9 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 Acknowledgements 26 4 References 264 Appendix 26 7 Index 273

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